CLS-08267; No of Pages 11 Cellular Signalling xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

Cellular Signalling journal homepage: www.elsevier.com/locate/cellsig

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Yuan Peng a,b,1, Lin Li a,c,1, Mengge Huang d, Changzhu Duan a,c, Luyu Zhang b, Junxia Chen a,c,⁎

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Article history: Received 31 July 2014 Accepted 17 August 2014 Available online xxxx

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Keywords: Angiogenin Ribonuclease inhibitor Interaction PI3K/AKT/mTOR Bladder cancer cells

Molecular Medicine and Cancer Research Center, Chongqing Medical University, Chongqing 400016, PR China The First Clinical College, Chongqing Medical University, Chongqing 400016, PR China Department of Cell Biology and Genetics, Chongqing Medical University, Chongqing 400016, PR China d Department of Clinical Medicine, Luzhou Medical College, Luzhou 646000, PR China

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Angiogenin interacts with ribonuclease inhibitor regulating PI3K/AKT/ mTOR signaling pathway in bladder cancer cells

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Angiogenin (ANG), a member of RNase A superfamily, is the only angiogenic factor that possesses ribonucleolytic activity. Recent studies showed that the expression of ANG was elevated in various types of cancers. Accumulating evidence indicates that ANG plays an essential role in cancer progression by stimulating both cancer cell proliferation and tumor angiogenesis. Human ribonuclease inhibitor (RI), a cytoplasmic protein, is constructed almost entirely of leucine rich repeats (LRRs), which are present in a large family of proteins that are distinguished by their display of vast surface areas to foster protein–protein interactions. RI might be involved in unknown biological effects except inhibiting RNase A activity. The experiment demonstrated that RI also could suppress activity of angiogenin (ANG) through closely combining with it in vitro. PI3K/AKT/mTOR signaling pathway exerts a key role in cell growth, survival, proliferation, apoptosis and angiogenesis. We recently reported that up-regulating RI inhibited the growth and induced apoptosis of murine melanoma cells through repression of angiogenin and PI3K/AKT signaling pathway. However, ANG receptors have not yet been identified to date, its related signal transduction pathways are not fully clear and underlying interacting mechanisms between RI and ANG remain largely unknown. Therefore, we hypothesize that RI might combine with intracellular ANG to block its nuclear translocation and regulate PI3K/AKT/mTOR signaling pathway to inhibit biological functions of ANG. Here, we reported for the first time that ANG could interact with RI endogenously and exogenously by using coimmunoprecipitation (Co-IP) and GST pull-down. Furthermore, we observed the colocalization of ANG and RI in cells with immunofluorescence staining under laser confocal microscope. Moreover, through fluorescence resonance energy transfer (FRET) assay, we further confirmed that these two proteins have a physical interaction in living cells. Subsequently, we demonstrated that up-regulating ANG including ANG His37Ala mutant obviously decreased RI expression and activated phosphorylation of key downstream target molecules of PI3K/AKT/mTOR signaling pathway. Finally, up-regulating ANG led to the promotion of tumor angiogenesis, tumorigenesis and metastasis in vivo. Taken together, our data provided a novel mechanism of ANG in regulating PI3K/AKT/mTOR signaling pathway via RI, which suggested a new therapeutic target for cancer therapy. © 2014 Published by Elsevier Inc.

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Angiogenin (ANG) is a 14 kDa angiogenic ribonuclease of the RNase A superfamily that is up-regulated in a variety of human cancers. It has a 33% amino acid identity and an overall homology of 56% to that of RNase-A [3]. ANG has been shown to undergo nuclear translocation in both cancer cells and cancer-associated endothelial cells where it binds to the promoter region of ribosomal DNA (rDNA) and stimulates ribosomal RNA (rRNA) transcription, which is necessary for angiogenesis induced by other angiogenic factors including VEGF, bFGF, aFGF, and EGF. ANG has been proposed as a permissive factor for angiogenesis [4,5]. Thus, ANG has a dual role in cancer progression; it stimulates cancer cell proliferation as well as mediates tumor angiogenesis, which suggested that ANG is a more effective molecular target for the development of cancer drugs [6,7].

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1. Introduction

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The bladder cancer is the most common tumor in the urinary system. Yearly almost 400,000 new cases of urinary bladder cancer are diagnosed in the world and more than 150,000 people die of the disease. In the US, bladder cancer is the fifth most frequent malignancy and the most expensive tumor to treat [1,2].

⁎ Corresponding author at: Department of Cell Biology and Genetics, Chongqing Medical University, No. 1, Yixueyuan Road, Chongqing 400016, PR China. Tel.: +86 23 68485806; fax: +86 23 68485555. E-mail address: [email protected] (J. Chen). 1 These authors contributed equally to this work.

http://dx.doi.org/10.1016/j.cellsig.2014.08.021 0898-6568/© 2014 Published by Elsevier Inc.

Please cite this article as: Y. Peng, et al., Angiogenin interacts with ribonuclease inhibitor regulating PI3K/AKT/mTOR signaling pathway in bladder cancer cells, Cellular Signalling (2014), http://dx.doi.org/10.1016/j.cellsig.2014.08.021

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T24 cells, pcDNA3.1(−)-myc-RI and pCMV-3 × FLAG-ANG plasmids were prepared by our laboratory. pECFP-N1 and pEYFP-N1 vectors were

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from Clontech. pGEX-4T-1 was obtained from GE Healthcare China. BALB/C nude (nu/nu) mice were purchased from Peking University Laboratory Animal Center (Beijing, PR China). The protocol was approved by The Ethics Committee of Chongqing Medical University. Mice were maintained according to the National Institutes of Health standards for the care and use of experimental animals. The care of laboratory animal and the animal experimental operation also conformed to Chongqing Administration Rule of Laboratory Animal. This housing facility is an ordinary housing facility, and it has in keeping with the national standard (Laboratory Animal—Requirements of Environment and Housing Facilities, GB 14925-2001). The animal production license and animal license of Chongqing Medical University are SYXK (Chongqing)2007-0001, SCXK(Chongqing)2007-0001. Fetal calf serum was from Hyclone (Logan, Utah, USA). DMEM/high glucose medium, RPMI 1640 medium and G418 were products of Gibco-BRL (Carlsbad, CA, USA). Lipofectamine 2000 reagent and TRIzol were bought from Invitrogen, Inc., (Carlsbad, California). Monoclonal primary mouse antibody of anti-human ANG was obtained from Abcam Biotechnology. Rabbit anti-human βactin and CD31 antibody were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Monoclonal primary rabbit antibody of antihuman PI3K, p-PI3K, PTEN, p-PTEN, Akt, GSK3(α/β), mTOR, p-Akt, pGSK3β, p-mTOR, and β-catenin were purchased from Bioworld Technology, Inc. (St. Louis, USA). The rest of the primary antibodies are from Beijing Zhongshang Biotechnology (Beijing, PR China).

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Human RI cDNA sequence (accession number: NM_002939) and human ANG cDNA (accession number: NM_001097577) were provided by the GenBank. Wild type (WT) ANG cDNA was located in the pCMV3 × FLAG vector. The following oligonucleotides were used to generate site-directed mutations: histidine 37 to alanine (H37A) forward, 5′-C ACA CAC TTC CTG ACC CAG GCC TAT GAT GCC AAA CCA CAG-3′, and reverse, 5′-CTG TGG TTT GGC ATC ATA GGC CTG GGT CAG GAA GTG TGT G-3′; the mutant H37A was generated by successive PCR reactions with the H37A primers. The pECFP-RI, pEYFP-ANG, and pGEX-4T-RI expression plasmids were generated using standard recombination DNA technique. The primers for pECFP-N1 plasmid (Clontech) were designed as follows: forward,5′-CCCAAGCTTATGAGCCTGGACATCCAG-3′,and reverse,5′CGGGATCCATGGAGATGACCCTCAGGGA-3′; the primers for pEYFP-N1 plasmid (Clontech) is the following: forward,5′-CCCAAGCTTATGGTGA TGGGCCTGGGCGT-3′,and reverse, 5′-CGGGATCCATCGGACGACGGAA AATTGAC-3′; and the pGEX-4T-1 vector (GE Healthcare) was used in the following primer pair: 5′-CGG GAT CCA TGA GCC TGG ACA TCC AGA G-3′,5′-CCG CTC GAG TCA GGA GAT GAC CCT CAG G-3′, annealed double-stranded oligonucleotides were inserted into the eukaryotic expression plasmids with DNA recombinant techniques. The recombinant plasmids pECFP-RI and pEYFP-ANG were identified by endonuclease HindIII and BamHI (underlined) digesting, and the pGEX-4T-RI was identified by endonuclease Xho I and BamHI (underlined) digesting. Finally they were further verified by DNA sequencing. All plasmids were verified by nucleic acid sequencing; subsequent analysis was performed using BLAST software (available on the World Wide Web at http:// www.ncbi.nlm.nih.gov/blast/).

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The T24 cells were grown in RPMI 1640 media with 10% fetal bovine serum in a humidified atmosphere containing 5% CO2 at 37 °C. The cells were seeded into six well plates, allowed to grow until 80–90% of the confluence, and then were transfected with the plasmids using Lipofectamine 2000 according to the manufacturer's protocols. Forty-eight hours after transfection, the selection was performed with 1000 μg/ml of G418 for 2 weeks and 500 μg/ml of G418 for additional 2 weeks. The individual G418-resistant monoclines were obtained by limiting

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ANG has been shown to activate AKT. AKT activation is known to upregulate ANG, also influence the PI3K, so there is a positive feedback loop between ANG and AKT [8,9]. It has been known that the production of ribosomal proteins is mediated by the AKT–mTOR pathway, and the production of rRNAs is likely mediated by ANG [3]. The PI3K/AKT/ mTOR signaling pathway is thought to be a central mediator in signal transduction pathways, which is frequently activated in diverse cancers [10]. Trouillon et al. reported that ANG activates nitric oxide synthase (NOS) by interacting with the cell nucleus. Similarly, NOS activity was stopped by blocking the PI3k/Akt kinase signaling transduction cascade, showing the importance of this pathway and ANG for NOS activity [11]. Kim et al. demonstrated that ANG induces transient phosphorylation of protein kinase B/Akt in cultured human umbilical vein endothelial (HUVE) cells [9]. The experiments suggest that cross-talk between ANG and protein kinase B/Akt signaling pathways could be essential for ANG-induced angiogenesis in vitro and in vivo [3,12,13]. However, the signal transduction pathway triggered by ANG-receptor interaction is currently unclear as the identity of a functional ANG receptor has not been fully determined. The mechanisms of ANG in cancer proliferation and angiogenesis remain largely unknown so far. Human ribonuclease inhibitor (RI), a cytoplasmic acidic protein with molecular weights of 50 kDa, contains 32 cysteine residues and consists of 15 leucine-rich repeats (LRRs). Such repeats have been identified in more than 100 proteins that exhibit a wide range of functions, including cell-cycle regulation, DNA repair, extracellular matrix interaction, and enzyme inhibition [14]. These leucine-rich repeats (LRRs) are present in a large family of proteins that are distinguished by their display of vast surface areas to foster protein–protein interactions. The unique structure and function of RI have resulted in its emergence as the central protein in the study of LRRs [15,16]. RI inhibits the great majority of the extracellular RNases from the vertebrate superfamily. The structure of RI, free or complexed to an RNase, has been determined. However, the physiological role of this protein has not been conclusively defined [17,18]. Therefore, we presume that RI could also possess unknown biological functions. The experiments have showed that RI might effectively inhibit tumor-induced angiogenesis [19,20]. Yet, a role for RI in angiogenesis is not fully clear in vivo [21,22]. In the post-genome era, protein–protein interactions play a central role in the life activity of the cells. ANG and RNase A have a highly conserved homologous sequence. Analysis of the sequence, structure and enzyme indicated that RI also might combine with ANG [23,24]. However, these experiments are not RI and ANG of the original position in vivo, whether endogenous intracellular RI can directly bind to ANG and associated biological mechanisms remain unclear so far. We recently reported that up-regulating RI inhibited the growth and induced apoptosis of murine melanoma cells through repression of angiogenin and PI3K/AKT signaling pathway [25]. The purposes of this study are to further reveal the molecular mechanism underlying interaction between ANG and RI and to provide new molecule target for bladder cancer therapeutics. Here, we assayed the interaction of RI and ANG using Co-IP, GST pull-down, FRET and laser scanning confocal microscopy. We present directed evidence for the first time that there is an endogenous and an exogenous interaction between RI and ANG in the cells. We found that up-regulating ANG including its His37Ala mutant promoted bladder tumor angiogenesis as well as tumor growth and metastasis through PI3K/AKT/mTOR signaling pathway. Therefore, data suggest that RI might combine with intracellular ANG to regulatePI3K/AKT/mTOR signaling pathway and nuclear translocation to affect its function. Our study may be of biological and clinical importance.

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Please cite this article as: Y. Peng, et al., Angiogenin interacts with ribonuclease inhibitor regulating PI3K/AKT/mTOR signaling pathway in bladder cancer cells, Cellular Signalling (2014), http://dx.doi.org/10.1016/j.cellsig.2014.08.021

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2.5. Western blot assay

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Cell total proteins were extracted using cell lysis buffer; total protein concentration was measured using Enhanced BCA Protein Assay Kit, andequal amounts (30 μg) of protein were loaded into each lane. The samples were separated by the electrophoresis in a 10% SDS-PAGE gel, and then were electrotransferred to a PVDF membrane at 200 mA for 1.5 h. Next, the samples were blocked with 5% skimmed milk powder in TBST buffer (0.1% Tween 20, 150 Mm NaCl, and 10 mmol/l Tris–HCl, pH 7.6) overnight at 4 °C. The membrane was probed with the primary antibody of RI (1:300 dilution), ANG (1:100 dilution), PI3K(1:500 dilution), p-PI3K(1:500 dilution), PTEN(1:500 dilution), p-PTEN(1:500 dilution), mTOR(1:500 dilution), p-mTOR(1:500 dilution), Akt (1:600 dilution), p-Akt (S473) (1:750 dilution), GSK3(α/β) (1:500 dilution), p-GSK3β (S9) (1:750 dilution), β-catenin(1:750 dilution) and β-actin (1:1000 dilution, actin of the same sample was used as an internal control) respectively for 2 h at 37 °C, washed thoroughly 3 × 10 min with TBST, and incubated with secondary antibody (1:2000 dilution)

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Approximately 500 μg of protein extracts was prepared from T24 262 cells that were transfected with pcDNA3.1(−)-myc-RI plasmid and 263 pCMV-3× FLAG-ANG plasmid for co-immunoprecipitation, then they 264

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Cells were seeded on cover slips in 6-well plates for 24 h, washed with phosphate-buffered saline (PBS; 3 × 5 min), fixed by cold 80% acetone for 20 min and then washed again with PBS (3 × 5 min). Cells were incubated with 3% bovine serum albumin (BSA) in PBS for 30 min at room temperature to block non-specific antibodies and then incubated with antibodies raised against RI (1:200), p-GSK3β (1:100), p-Akt (1:100), p-mTOR(1:100),or ANG(1:200) overnight at 4 °C. After being washed three times with PBS, cells were incubated with secondary antibody, namely Alexa Fluor 488 goat anti-mouse IgG (1:100 dilution) or Alexa Fluor 594 goat anti-rabbit IgG (1:100 dilution), for 1 h at 37 ° C in the dark and washed three times. Cell nuclei were stained with 4, 6-diamidino-2-phenylindole (DAPI). Cells were washed again with PBS (3 × 5 min) and finally sealed with 50% glycerin. Observations were performed under an Olympus multifunction microscope (Tokyo, Japan). Each assay was performed in triplicate. For the colocalization assay of RI and ANG, the cells were incubated for 24 h on cover slips in 6-well plates, the cells were fixed with 4% paraform for 30 min, then washed and blocked with 3% BSA for 30 min in PBS For 30 min, Then they were incubated with ANG and RI primary antibodies, and were incubated with secondary antibody Alexa Fluor 594 goat antirabbit IgG (1:100 dilution) and Alexa Fluor 488 goat anti-mouse IgG (1:100 dilution) for 1 h at 37 °C in the dark, then washed each thrice with PBS for 5 min. Cell nuclei were stained with 4,6-diamidino-2phenylindole (DAPI), finally washed again with PBS (3 × 5 min) and sealed with 50% glycerin. Observations were performed under a laser scanning confocal microscope (Leica TCS-SP2, German).

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The total cellular RNA was isolated from various T24 cells using the TRIzol reagent according to manufacturer's instruction. Reverse transcription was performed and cDNAs were amplified with the following primer pairs: ANG forward: 5′-CAG CAC TAT GAT GCC AAA CCA C-3′ and reverse: 5′-GAA ATG GAA GGC AAG GAC AGC-3′;GAPDH of the same sample was used as an internal control, GAPDH forward:5′-AGA AGG CTG GGG CTC ATT TG-3′ and reverse: 5′-AGG GGC CAT CCA CAG TCT TC-3′. RT-PCR was conducted with the following parameters: 37 °C for 15 min, 85 °C for 5 s for RT reaction, then 94 °C for 2 min, 94 °C for 30 s, 56 °C for 30 s and 72 °C for 30 s and a total of 30 cycles, then a final extension of 72 °C for 10 min. The PCR products were analyzed by electrophoresis on a 1% agarose gel, stained with GoldView and photographed under UV illumination. Results were collected and analyzed with MJ Opticon Monitor Analysis Software (Bio-Rad). Experiments were performed in triplicate and repeated three times.

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respectively for 1 h at 37 °C, washed thoroughly 3 × 10 min with TBST. Blots with immuno-complexes were detected by enhanced chemiluminescence method (Thermo Scientific). The results were collected and analyzed with MJ Opticon Monitor Analysis Software (BioRad). Experiments were performed in triplicate.

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dilution, then proliferated and expanded to generate stable transfected cell lines that express ANG and the blank control vector, and named T24-ANG, T24-ANGH37A cell lines and T24 vector cell lines, respectively.

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Fig. 1. ANG and site-directed mutant of ANG expression are evaluated by RT-PCR, Western blot and immunofluorescence after transfection for 48 h. (A) ANG and ANGH37A mRNA expression levels were analyzed with RT-PCR in T24 cells. M: DNA maker; lanes 1, 3, 5, 7: GAPDH; lanes 2, 4, 6, 8: ANG; lanes 1, 2: T24 cells, lanes 3, 4: T24 vector cells, lanes 5, 6: T24-ANG cells, lanes 7, 8: T24-ANGH37A cells. RT-PCR of ANG was a product of 400 bp and GAPDH was a product of 330 bp as internal control. (B) Immunofluorescence observation of ANG and ANGH37A was further detected. T24-ANG cells and T24- ANGH37A cells showed remarkably stronger immunofluorescence signal in cytoplasm compared with the other control cells respectively (magnification ×200). (C) and (D) ANG and RI protein expression level was determined with Western blot. Proteins were separated by 10% SDS-PAGE and analyzed with monoclonal ANG antibody or polyclonal RI antibody. β-Actin was used as internal control. The levels of ANG proteins were obviously increased by transfected with pCMV-3 × FLAG-ANG or pCMV3 × FLAG-ANGH37A compared with the control groups.

Please cite this article as: Y. Peng, et al., Angiogenin interacts with ribonuclease inhibitor regulating PI3K/AKT/mTOR signaling pathway in bladder cancer cells, Cellular Signalling (2014), http://dx.doi.org/10.1016/j.cellsig.2014.08.021

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GST-RI fusion proteins were purified using Glutathione-Sepharose 4B (GE Healthcare, Little Chalfont, UK) according to the manufacturer's instructions. Purified GST fusion proteins were then incubated with equal amounts of FLAG-ANG (transfected HEK293 cell extracts) or purified FLAG-UHRF2 (anti-FLAG M2 affinity gel) for 2 h at 4 °C with constant shaking. Glutathione-Sepharose 4B beads were washed with icecold phosphate-buffered saline (pH 7.4) for 3 times. Bound proteins

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2.9. Confocal microscope scanning and fluorescent resonance energy transfer 283 (FRET) analysis 284 293 and T24 cells were co-transfected with pECFP-RI and EYFP-ANG or pEYFP-ANGH37A Twenty-four hours after transfection, 293 and T24 cells were examined by confocal microscope with a 40 × oil immersion objective. Confocal microscopic images were obtained with a Leica confocal imaging spectrophotometer system (TCS-SP, Germany). An excitation wavelength of 458 nm and an emission wavelength of 470–500 nm were used for CFP, and an excitation wavelength of 514 nm and an emission wave-length of 520–550 nm were used for yellow fluorescent protein (YFP). For assessing FRET between the two fluorophores, an acceptor photobleaching method using the laser-scanning confocal

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were eluted by elution buffer (50 mM Tris–Cl, 10 mM reduced glutathi- 280 one, pH 8.0); eluted proteins were subjected to SDS-PAGE analysis 281 using Coomassie brilliant blue staining and Western blot identification. 282

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were incubated with anti-Myc antibodies for 1 h, and added with 30 μl of protein A-agarose (Invitrogen) at 4 °C overnight. After they were washed 5 times with the IP buffer, bound proteins were released by boiling in 20 μl 2 × SDS loading buffer for 3 min. Released proteins were examined by Western blotting with anti-RI or anti-ANG antibodies, using the ECL chemiluminescent system (Amersham Biosciences). Each assay was performed in triplicate.

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Fig. 2. RI interacts with ANG or ANGH37A in vivo and in vitro. The interaction of RI with ANG was examined as described in the “Materials and methods” section with GST pull-down and coimmunoprecipitation (Co-IP). (A) The bacteria were induced with 0.1 mmol/l IPTG in different times, and then were broken by ultrasonic. The aim protein band was identified by 10% SDSPAGE and Coomassie brilliant blue staining. GST and GST-RI fusion proteins were at the relative molecular weight of 26 × 103 and 76 × 103 locations respectively after sepharose 4B gel purification. (B) and (C) Western blotting results showed purified GST-RI fusion protein and GST appeared as specific reaction bands at the relative molecular weight of 26 × 103 and 76 × 103 locations respectively by the RI and GST antibody incubation. (D) and (E) Western blot displayed that ANG protein of the cell lysate from transfected pCMV-3 × FLAGANG or pCMV-3 × FLAG-ANGH37A could be combined with GST-RI and be specifically pulled down, indicating that RI could specifically bind to ANG or ANGH37A in vitro. (F) and (G) RI was co-immunoprecipitated with ANG in 293 and T24 cells. The cell lysates were incubated with anti-Myc antibody, and the immunoprecipitates were subjected to immunoblot by anti-ANG and anti-RI antibodies respectively. The expression of FLAG-ANG only could be detected in Myc–RI interacting protein complexes with anti-myc antibody precipitation, which suggest that RI might interact with ANG in cells.

Please cite this article as: Y. Peng, et al., Angiogenin interacts with ribonuclease inhibitor regulating PI3K/AKT/mTOR signaling pathway in bladder cancer cells, Cellular Signalling (2014), http://dx.doi.org/10.1016/j.cellsig.2014.08.021

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Fig. 3. Fluorescent resonance energy transfer and colocalization of ANG with RI are assayed. (A–D) 293 and T24cells were co-transfected with plasmids as indicated and subjected to photobleaching analysis. The CFP and YFP fluorescence emission intensities in the cells before and after photobleaching were shown. (E) The FRET efficiency (FRETe) was calculated as the percentage of the CFP fluorescence recovery. All the data represented the means of three independent experiments with the bars showing standard deviation. (F) RI expression and colocalization assay with laser scanning confocal microscope. Immunofluorescence staining of RI (red) and ANG (green) in four kinds of T24 cells. The nucleus was counterstained with DAPI (blue). The merge images (yellow) demonstrated that ANG or ANGH37A with RI is colocalized in cells. The expression of RI markedly decreased in transfected ANG or ANGH37A cells compared with the control group cells (magnification ×200).

Please cite this article as: Y. Peng, et al., Angiogenin interacts with ribonuclease inhibitor regulating PI3K/AKT/mTOR signaling pathway in bladder cancer cells, Cellular Signalling (2014), http://dx.doi.org/10.1016/j.cellsig.2014.08.021

Y. Peng et al. / Cellular Signalling xxx (2014) xxx–xxx

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Fig. 4. Up-regulating ANG and ANGH37A promote the expressions of PI3K/AKT/mTOR signaling pathway molecules in vitro. (A–D) The cells' total proteins were extracted using cell lysis buffer; the protein expressions of PI3K, p-PI3K, PTEN, p-PTEN, AKT, p-AKT (S473), GSK3 (α/β), p-GSK3β (S9), mTOR, p-mTOR and β-catenin were examined using corresponding antibodies by immunoblot analysis. The results showed that transfections with pCMV-3 × FLAG-ANG or pCMV-3 × FLAG-ANGH37A in T24 cells remarkably up-regulated the expressions of p-PI3K, p-PTEN, p-Akt (S473), p-GSK3β (S9), p-mTOR and β-catenin compared with control group cells respectively, but the protein expressions of AKT, PI3K, mTOR and GSK3 (α/β) did not change obviously, compared with the control group cells respectively. β-Actin served as an internal control for sample loading. Relative protein levels were normalized against those of β-actin by MJ Opticon Monitor Analysis Software (Bio-Rad), data were expressed as means ± S.D. (n = 3).*p b 0.05. (E) Immunofluorescence assay of RI, p-Akt, p-GSK3β and p-mTOR in cells. Immunofluorescence staining as described in the “Materials and methods” section. The expression of RI in T24-ANG and T24-ANGH37A cell groups showed obviously weaker immunofluorescence signal in cytoplasm, whereas, the expression of p-Akt, p-GSK3β and p-mTOR were stronger in T24-ANG and T24-ANGH37A cell groups, compared with the other control cells respectively (magnification ×200).

Please cite this article as: Y. Peng, et al., Angiogenin interacts with ribonuclease inhibitor regulating PI3K/AKT/mTOR signaling pathway in bladder cancer cells, Cellular Signalling (2014), http://dx.doi.org/10.1016/j.cellsig.2014.08.021

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2.10. Tumor growth model

2.12. Statistical analysis

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Four kinds of single cell suspension of 0.1 ml containing 2 × 10 tumor cells including the T24-ANGH37A cells, the T24-ANG cells, the T24 vector cells, and the T24 cells, were respectively injected into the left flank of the syngeneic BALB/c nude mice (8–12 weeks old, SPF degree, 20 ± 3 g). Each group contained eight mice. The time of tumor formation was recorded; 4 weeks after injection, the mice were sacrificed. The tumors were removed and weighed. The promoting rates of tumors were calculated by the following formula: promoting rate = (tumor weight of treatment group − tumor weight of control vector group) / tumor weight of treatment group × 100%. Then, a part of the tumors tissue was fixed with formalin for pathology analysis, the others were quickly placed in liquid nitrogen for the frozen section of immunofluorescence assay.

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vessels. Immunohistochemistry and immunofluorescence assays for RI, ANG, p-mTOR, p-Akt and p-GSK3β were done to detect the impact of ANG and RI on angiogenesis as well as PI3K/AKT/mTOR signaling pathway. The antigen retrieving steps were taken in the citrate buffer for 10 min at 95–98 °C in the microwave oven. Tissue sections were incubated overnight in primary antibodies of RI (1:200 dilution), βcatenin (1:100 dilution) and ANG (1:100 dilution) at 4 °C respectively, then incubated in secondary antibodies (IgG/Bio, Ready to use) for 15 min at 37 °C, using SP (Streptavidin/Peroxidase) HistostainTM-Plus Kits, DAB staining. The nuclei were counterstained by hematoxylin. The rest of the procedure was performed according to manufacturer's instruction. As mentioned above in the immunofluorescence assays, the tumor frozen tissue sections were incubated in monoclonal rabbit primary antibodies of p-Akt (1:100 dilution), p-GSK3β (1:100 dilution), ANG (1:100 dilution) and CD31 (1:50 dilution), overnight at 4 °C. After that they were washed three times with PBS, incubated with Fluorescein-conjugated Goat Anti-rabbit IgG (1:100 dilution) for 1 h at 37 °C in the dark, washed thrice with PBS for 5 min, and finally sealed with 50% glycerin. Observations were performed using an Olympus multifunction microscope (Tokyo, Japan).

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ANG eukaryotic expression vectors and site-directed mutant 353 ANGH37A were constructed and identified with endonuclease digesting 354 and DNA sequencing; two bands, about 444 bp and 6300 bp were 355

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The tumor tissue were fixed in 10% buffered formalin and embedded in paraffin, and 5 μm sections were stained with HE (hematoxylin– eosin). The microvessels were counted from 10 different fields under microscope (×200) corresponding to areas with the highest density of

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3.1. ANG, mutant ANGH37A eukaryotic expression plasmid and transfection 351 efficiency are identified 352

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All data were dealt with SPSS18.0 statistical software. The values 347 were expressed as means ± SD. Student's t tests were used for statistical 348 analysis. p values of b0.05 were considered to be significant. 349 3. Results

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microscopy was applied. CFP and YFP fluorescence images were collected repetitively at a 10 s interval for five times. After that, YFP was photobleached with the full power of the 514 nm line laser, and the emission intensities of CFP and YFP were monitored for an additional five times. FRET efficiency (FRETE) was calculated as the percentage of the CFP fluorescence recovery [FRETE = (CFPA − CFPB) / CFPA × 100%], where CFPA was the CFP intensity after photobleaching and CFPB was the CFP intensity before photobleaching of YFP. FRETE should be no more than 0 in the absence of FRET. Otherwise, FRET is considered to have occurred. Photobleaching data were from 10 independent experiments.

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Fig. 5. Up-regulating ANG or ANGH37A promotes tumor growth and spontaneous lung metastasis of BALB/c nude mouse xenograft model. 2 × 105 tumor cells including the T24 cells, the T24 vector cells, the T24-ANG cells and T24-ANGH37A cells were respectively injected subcutaneously into the backs of the BALB/c nude mice. After 4 weeks, the mice were sacrificed. The tumors and lungs were isolated, weighed, and photographed. (A) Representative images of the tumor bearing BALB/c nude mice and xenograft tumor. (B) Tumor weight analysis. The T24ANG and T24-ANGH37A cell groups significantly promoted the growth of xenograft tumor compared with control groups. (C) HE staining of lung sections, arrows show invasive tumor cells in lung. The experiment showed that ANG increased the metastasis of bladder cancer cells with more metastasis nodules and invasive tumor cells. (D) Immunofluorescence staining with an antibody against CD31 antigen of vascular endothelial cells. (E) HE staining of tumor sections, arrows indicate microvessels among the tumors. (F) Microvessel density analysis (magnification 200×). Immunofluorescent and histochemical study demonstrated that numerous microvessels could be seen among the tumors of the mice injected with T24-ANG cells and T24-ANGH37A cells. In contrast, there were a few microvessels in the tumor of the mice injected with T24 or T24 vector cells (magnification ×200).

Please cite this article as: Y. Peng, et al., Angiogenin interacts with ribonuclease inhibitor regulating PI3K/AKT/mTOR signaling pathway in bladder cancer cells, Cellular Signalling (2014), http://dx.doi.org/10.1016/j.cellsig.2014.08.021

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Fig. 6. Up-regulating ANG and ANGH37A promote the expressions of PI3K/AKT/mTOR signaling pathway molecules in vivo. (A) Immunofluorescence sections were stained in tumor xenograft tissue with mouse anti-human ANG, rabbit anti-human RI, p-Akt (S473), p-GSK3β (S9) and p-mTOR respectively, then they were incubated with Alexa Fluor 594 Goat Anti-Rabbit IgG or Alexa Fluor 488 Goat Anti-Mouse IgG secondary antibody. T24-ANG and T24-ANGH37A cell groups resulted in higher p-Akt (S473), p-GSK3β (S9) and p-mTOR as well as lower RI expressions in tumor tissue, compared with T24 and T24 vector cell groups, which is consistent with experiment in vitro (magnification ×200). (B) Immunohistochemistry sections were stained in tumor xenograft tissue with primary antibodies of ANG, p-AKT, p-GSK3β and p-mTOR respectively, the nuclei were counterstained by hematoxylin (200 ×magnification). The experiment showed that the T24ANG and T24-ANGH37A cell groups had higher brown staining of ANG as well as stronger immunostain of p-AKT, p-GSK3β and p-mTOR in cytoplasm compared with control cell groups (magnification ×200).

Please cite this article as: Y. Peng, et al., Angiogenin interacts with ribonuclease inhibitor regulating PI3K/AKT/mTOR signaling pathway in bladder cancer cells, Cellular Signalling (2014), http://dx.doi.org/10.1016/j.cellsig.2014.08.021

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3.3. Fluorescent resonance energy transfer and colocalization of ANG with RI are determined

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We then applied the FRET technique to detect ANG-RI interaction. After co-transfecting 293 and T24 cells with pECFP-RI and pEYFP-ANG or pEYFP-ANGH37A, the energy acceptor was photobleached by a highintensity exposure to the YFP excitation light (514 nm laser) to block energy transfer from CFP to YFP, and the energy redistribution of CFP was recorded. If there is a FRET, photobleaching of YFP should increase the emission of CFP at 488 nm. As shown in Fig. 3A–E, the FRET did occur between ANG and RI, further confirming that ANG interacts with RI in living cells. A presumption that RI and ANG are functional partners implies that they would localize to the same cellular compartment. In order to further investigate whether RI interacts with ANG, the subcellular localization of endogenous ANG and RI were detected in 293 and T24 cells by immunofluorescence microscopy. The immunofluorescence study revealed that native ANG exhibited both in cytoplasm and nucleus (green). ANG, as a nuclear translocation protein, appeared as many spots in the nucleus and was scattered in cytoplasm. Most of the distribution of RI showed a cytoplasmic diffuse staining while other small

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In order to explore the molecular mechanism of the promotion of cell proliferation after up-regulating ANG, we determined the protein levels of PI3K and the important signaling pathway target molecules of PI3K/AKT/mTOR by Western blot. Then, the semiquantitative analysis of the protein levels of PI3K, p-PI3K, Akt, p-Akt, GSK3 (α/β), p-GSK3β, PTEN, p-PTEN, mTOR, p-mTOR and β-catenin were performed. The protein levels of PI3K, AKT, mTOR and GSK3 (α/β) did not change obviously. However, The protein levels of p-mTOR, p-Akt, p-PI3K, p-GSK3β and β-catenin were increased by 121.62%, 77.76%, 68.00%, 63.64% and 41.82% respectively in T24-ANG as well as 115.42%, 77.79%, 68.54%, 63.64% and 48.57% respectively in T24-ANGH37A cell group (*p b 0.05, **p b 0.01) (Fig. 4A–D). Furthermore, PTEN expressions were obviously decreased in T24-ANG and T24-ANGH37A cell groups, whereas phosphorylation of PTEN was up-regulating in T24-ANG and T24-ANGH37A cell groups, compared with control groups. Immunofluorescent analysis revealed that p-Akt, p-GSK3β and p-mTOR were obviously induced as well as RI was significantly weaker in T24-ANG and T24-ANGH37A cell groups compared with the other control groups respectively (Fig. 4E). The results were consistent with Western blot, which suggest that the up-regulating ANG might be associated with the activation of the PI3K/AKT/mTOR signaling pathway.

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To further verify the interaction, we performed in vitro GST pulldown and in vivo co-immunoprecipitation assay. The bacteria were induced with 0.1 mmol/l IPTG in different times, and then were broken by ultrasonic; crushing product was identified by 10% SDS-PAGE. The aim protein band was visible at the relative molecular weight of 76 × 103 location (higher than 72 × 103 maker), and the fusion protein expression was higher under the induction of 0.1 mmol/l IPTG concentration for 3 h and 5 h (Fig. 2A). Coomassie brilliant blue staining and Western blotting showed that purified GST and GST-RI fusion proteins appeared as specific bands at the relative molecular weight of 26 × 103 and 76 × 103 locations (Fig. 2B and C), which is consistent with our expectations. Western blot displayed that ANG protein of the cell lysate from transfected pCMV-3 × FLAG-ANG or pCMV-3 × FLAG-ANGH37A could be combined with GST-RI and be specifically pulled down, indicating a directly physical interaction between ANG and RI in vitro (Fig. 2D and E). In order to further study the interaction between RI and ANG in the cell, we performed co-immunoprecipitation assay. The pcDNA3.1-mycRI and pCMV-3× FLAG-ANG were co-transfected into 293 cells and T24 cells, broken cell lysates were immunoprecipitated with anti-Myc antibody, followed by Western blot with anti-RI and anti-ANG antibodies respectively. The results showed only the precipitation of Myc-RI interacting protein complexes from co-transfected cells with the pcDNA3.1-myc-RI and pCMV-3 × FLAG-ANG, could be detected the expression of FLAG-ANG, indicating the presence of the interaction between RI and ANG in the cellular content (Fig. 2F and G).

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part showed nuclear distribution (red). More significantly, the merge figure could be observed with a yellow area, which showed that there was a colocalization of ANG with RI. Meanwhile, the expression of RI was decreased notably in the T24-ANG or T24-ANGH37A cell group compared with the other two control groups (Fig. 3F).

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generated after HindIII and BamHI digestion (Supplementary Figs. S1A and S2A). The DNA sequencing results also indicated identical nucleotide sequence with the design (data not shown), which confirmed the correct construction of plasmids. 48 h after transfection, the transfected cells were isolated by G418 selection, and then cloned, expanded, finally identified by RT-PCR (Fig. 1A), immunofluorescence assay (Fig. 1B) and Western blot (Fig. 1C and D). The expression of ANG mRNA obviously increased by 136% or 153% in T24-ANG cells and T24-ANGH37A cell and protein levels were significantly heightened by 271% or 255% in T24ANG cell group and T24-ANGH37A cell group, compared with the other two control groups respectively. Immunofluorescence analysis also showed that ANG was significantly stronger in T24-ANG cellgroup and T24-ANGH37A cell group, compared with the control cell group. The results indicated that ANG had stably high expressions in T24-ANG cells and T24-ANGH37A cells.

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3.5. ANG overexpression promotes tumor growth and spontaneous lung 480 metastasis of BALB/c nude mice 481 To further affirm the influence of the ANG on tumor growth, 2 × 105 various kinds of T24 cells in 0.1 ml of PBS were implanted subcutaneously into the left flank of mice. The results showed that the T24-ANG and T24-ANGH37A cell groups significantly promoted the growth of bladder cancer compared with the other control groups. The tumors in the T24-ANG and T24-ANGH37A cell groups were bigger than those in the other control groups (Fig. 5A and B). The promoting rates of tumors were 46.67% in T24-ANG cell group and 48.94% in T24-ANGH37A cell group compared with T24 vector cell group, respectively, **p b 0.01 (Fig. 5A and B). Mice injected with T24-ANG and T24-ANGH37A cell groups also showed a significant increase of the spontaneous lung metastasis, compared with T24 and T24 vector cell groups (Fig. 5C). To verify whether ANG and RI are correlated with the new blood vessel formation through PI3K/AKT/mTOR signaling pathway, pathological and immunofluorescent analyses of CD31 were implemented. The results showed that the T24-ANG and T24-ANGH37A cell groups displayed a high CD31expression and apparent increase of angiogenesis in tumor tissue; whereas weaker CD31 expression and less microvessels were seen in the tumor tissue of the control groups (Fig. 5D). Microvessels were counted from 10 different fields under a microscope (magnification ×200) corresponding to areas with the highest density of vessels on the HE sections. T24-ANG and T24-ANGH37A cell groups showed more vessels compared to the other control groups (**p b 0.01) (Fig. 5E and F).

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3.6. ANG overexpression increases expressions of PI3K/AKT/mTOR signaling 506 target molecules in vivo 507 Immunohistochemistry and immunofluorescence assays were done to further confirm ANG expression and its impact on RI and PI3K/AKT/ mTOR signaling pathway in tumors. The results showed that T24-ANG and T24-ANGH37A cell groups revealed much lower RI expression as

Please cite this article as: Y. Peng, et al., Angiogenin interacts with ribonuclease inhibitor regulating PI3K/AKT/mTOR signaling pathway in bladder cancer cells, Cellular Signalling (2014), http://dx.doi.org/10.1016/j.cellsig.2014.08.021

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Ribonuclease inhibitor (RI) is an important intracellular protein with many functions. RI is constructed almost entirely of leucine-rich repeats, which might be involved in some unknown biological functions like 522 other structurally similar proteins. The unique structure and function 523 of RI have resulted in its emergence as the central protein in the study 524 of LRRs [26,27]. Our previous works suggest that RI might have tumor 525 suppressor gene function inhibiting some tumor growth, metastasis 526 and angiogenesis [28–30]. Recent research showed that angiogenin 527 (ANG) could effectively promote angiogenesis and tumor cell prolifera528 tion, which ANG is one of the most potent angiogenic factors in vivo. 529 ANG might stimulate ribosomal RNA transcription and the ribosome 530 biogenesis through nuclear translocation and PI3K/Akt/mTOR signaling 531 pathway. The dual role of ANG in cancer progression suggested that 532 ANG is a molecular target for the development of cancer drugs [3,31]. 533 Q9 Betty et al. demonstrated that ACTIBIND, a T2 RNase from the fungus As534 pergillus niger, competed with ANG and inhibited human melanoma 535 growth, angiogenesis, and metastasis. ANG antagonists including its 536 monoclonal antibody, a soluble binding protein, antisense oligos, a nu537 clear translocation blocker, and an enzymatic inhibitor have all been 538 shown to inhibit xenograft growth of human tumor cells in athymic 539 mice [32,33]. But, because these observations were from studies of cell 540 culture and xenograft tumor models, it is unknown whether ANG 541 plays a role in cancer initiation from its orthotopic origins and whether 542 it is involved in cancer progression in the natural environment. The role 543 of ANG in the growth and progression of tumors arising in a more nature 544 environment has not been studied before. The experiment demonstrat545 ed that RI could inhibit ANG activity by binding to it in vitro [34]. How546 ever, the underlying mechanism remains largely unknown to date. And 547 that, to our knowledge, there is almost no direct evidence of RI involving 548 in the crosstalk between ANG and PI3K/Akt/mTOR signaling pathway 549 Q10 that has been reported. 550 In the present study, we found that RI and ANG have a subcellular 551 colocalization and endogenous and exogenous interactions 552 between them with a laser scanning confocal microscope, co553 immunoprecipitation, GST pull-down and FRET. For the first time, we 554 got the direct evidence of interaction between RI and ANG in cells. The 555 amino acid sequence of ANG is highly conservative compared with 556 RNase A. In vitro studies showed that RI could also inhibit ANG activity 557 by extraordinarily tight combination. Binding to RI blocks the active 558 site of the enzyme and abolishes ribonucleolytic activity [35]. Artificial 559 complexes of RI and ANG inhibit the angiogenesis of chick chorioallanto560 ic membrane [36]. Kimberly et al. report that RI affects ANG-induced 561 neovascularization of rabbit corneas, which provide the first direct 562 Q11 data in that RI serves to regulate the biological activity of ANG [34]. 563 Our experiment results supported these findings. We speculate that 564 the RI might bind directly with intracellular ANG to block the active cen565 ter of ribonuclease of ANG and prevent the nuclear translocation of ANG, 566 which result in the inhibition of rRNA transcription promotion and other 567 functions of ANG. 568 Moreover, we showed that up-regulating ANG negatively regulated RI 569 expression in both cells and tissue, and that RI could combine and interact 570 with ANG, which is consistent with our recent report [25]. Further re571 search on its mechanism and the relationship of interaction between 572 them is being carried out in our laboratory. Furthermore, we also found 573 that up-regulation of ANG remarkably increased the phosphorylation of 574 signaling targets PI3K, AKT, GSK-3β and mTOR. It has been known 575 that PI3K/AKT/mTOR signaling pathway regulates cell proliferation,

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differentiation, cellular metabolism, apoptosis and cancer cell survival [37]. We previously demonstrated that down-regulation of ANG could inhibit the phosphorylation of Akt, GSK3β and mTOR in bladder cancer cells in vivo and vitro. Consistently, there is a crosstalk between AKT and ANG pathways. For example, ANG activates AKT, and activated AKT in turn stimulates nuclear translocation of ANG [38]. The production of ribosomal proteins is mediated by the AKT–mTOR pathway. mTOR plays an important role in PI3K- and AKT-dependent oncogenesis [3]. By promoting the phosphorylation of AKT, which is necessary for angiogenin-induced wound healing migration of HUVE cells and angiogenin-induced angiogenesis in chick chorioallantoic membrane [9]. The mTOR acts as a downstream effector for Akt, AKT can directly phosphorylate and activate mTOR. L Chang et al. demonstrated that activation of the PI3K/Akt/ mTOR signaling pathway resulted in cancer cell growth, survival, invasion, DNA repair and metastasis [39]. GSK-3β also plays a role in cell proliferation via regulation of genes involved in cell cycle progression and survival. Likewise, activated AKT also phosphorylate GSK-3 on ser9. Our experiment results agree with these findings. In addition, we also showed that phosphatase and tensin homolog deleted on chromosome ten (PTEN) expressions that were remarkably decreased in T24-ANG and T24-ANGH37A cell groups, whereas the phosphorylation of PTEN was up-regulating in T24-ANG and T24ANGH37A cell groups, compared with control groups. PTEN acts as a tumor suppressor gene through the action of its phosphatase protein product. This phosphatase is involved in the regulation of the cell cycle, preventing cells from growing and dividing too rapidly. It is mutated or deleted with high frequency in various human cancer tissues to promote tumorigenesis. Unlike most of the protein tyrosine phosphatases, this protein preferentially dephosphorylates phosphoinositide substrates. It negatively regulates intracellular levels of phosphatidylinositol-3,4,5trisphosphate (PIP3) in cells and functions as a tumor suppressor by negatively regulating Akt/PKB signaling pathway [40], which is consistent with our experiment results. In our study, we also detected the histidine-37 of human angiogenin, which is the active site of angiogenin. We constructed the site directed mutagenesis of histidine-37 of alanine to assay influence on the interaction between human ribonuclease inhibitor and the PI3K/AKT/mTOR signaling pathway. The results showed that the interaction between RI and ANGH37A did not change significantly in vivo and vitro, compared with the wild type ANG. We presumed that the active site may be not the interaction site of RI and ANG. However, the results also provided us a clue that the interaction between the two proteins might be not one site but a domain, and the following further research is being carried out in our laboratory by more mutations. Finally, the animal experiment showed that up-regulating ANG including ANG His37Ala mutant significantly promoted the tumor xenografts growth and metastasis; tumors in T24-ANG and T24-ANGH37A cell groups were larger and higher in microvascular density than those in control groups. Angiogenesis is necessary for the growth and metastasis of tumors. The growth of solid tumors depends on the induction of angiogenesis to provide adequate oxygen and nutrients to proliferating cells and avoid necrosis [41]. But, less metastasis of the lung was detected in control cell groups, moreover, T24-ANG and T24-ANGH37A cell groups showed a remarkable invasion and metastases. ANG is a prominent angiogenic factor that has been shown to have a dual effect on tumor progression by inducing both angiogenesis and cancer cell proliferation through stimulating ribosomal RNA transcription in both endothelial cells and cancer cells. Compelling evidence indicates that ANG activity is necessary for other angiogenic factors to induce angiogenesis. ANGinduced rRNA transcription appears to be a common down-stream event of tumor angiogenesis. Thus, ANG inhibitors have been shown to inhibit not only ANG-induced angiogenesis but also those induced by other angiogenic factors including VEGF, FGF, and EGF. ANG inhibitors would therefore have a profound effect in inhibiting tumor angiogenesis [42]. In conclusion, according to our work and reports, we put forward hypothesis: RI might regulate the function of ANG in two following ways

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well as stronger positive signal of p-Akt, p-GSK3β and p-mTOR expression in tumor tissue, whereas weaker p-AKT, p-GSK3β and p-mTOR expression were seen in tumor tissue of the control groups (Fig. 6A and B). These results were consistent with assays in vitro and strongly suggest that the up-regulating ANG may be associated with the promotion of PI3K/AKT/mTOR signaling pathway.

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This study was supported by grants from the National Natural Science Foundation of China (81172424 and 81372203).

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or one of them: RI could regulate the cross-talk between angiogenin and PI3K/AKT/mTOR; RI also could bind directly with intracellular ANG to the block active center of ribonuclease of ANG and prevent the nuclear translocation of ANG, which result in the inhibition of rRNA transcription promotion and other function of ANG. Further research on its mechanism and the relationship of interaction between them is being carried out in our laboratory. Taken together, these results might reveal a better understanding of the molecular mechanism underlying the role of ANG in cell proliferation and tumor growth. Our findings highlight the possibilities that ANG could serve as a biological marker and be a valuable target for the therapy of bladder cancer. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.cellsig.2014.08.021.

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