Materials
The antibodies and reagents used in this study were obtained from the following sources: anti-IRS-2 and anti-IGF-IR antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Anti-USP9X antibody was kindly provided by Dr. Stephen A. Wood (Griffith University, Queensland, Australia). Anti-USP9X antibody was purchased from Abcam (Cambridge, MA, USA). Anti-Akt, anti-pAkt (Ser473), anti-Erk1/2, anti-pErk1/2, and anti-IGF-IR antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-MMP-9 antibody was purchased from Cell Signaling Technology and Abcam. Pepstatin A was purchased from Sigma–Aldrich (St. Louis, MO, USA) and Pefabloc SC was obtained from Cayman Chemical (Ann Arbor, Michigan, USA). Gelatin and PD98059 were purchased from Sigma–Aldrich, and BMS754807 was purchased from Funakoshi (Tokyo, Japan).
Cell culture
Prostate cancer PC3 cells, which were kindly provided by from Dr. Akio Matsubara (Hiroshima University) were cultured as described previously36, which were kindly provided by Dr. Eijiro Nakamura (Kyoto University, Kyoto, Japan) were cultured as described previously37.
Immunoblotting analysis
Cells were serum-starved for 20–24 h in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 0.1% bovine serum albumin (BSA), and then incubated with or without human recombinant IGF-I (100 ng/ml) for 5 min. Cell lysates were prepared with lysis buffer (50 mM Tris-HCl, pH 7.4, 1 mM EDTA, 1 mM EGTA, 150 mM NaCl, 50 mM NaF, 1% Toriton X-100, 100 kallikrein-inactivating units/ml aprotinin, 20 mg/ml phenylmethanesulfonyl fluoride, 10 mg/ml leupeptin, 5 mg/ml pepstatin) and subjected to immunoprecipitation and immunoblotting as described previously36. The levels of each protein were quantified using ImageJ.
Preparation of conditioned medium
After cells were transfected with human IRS-2 small interfering RNA (siRNA) or control siRNA for 24 h, the medium was replaced with serum-free medium. After 24 h of serum starvation, the medium was collected, centrifuged to remove cell debris, and concentrated 20 times using centrifugal filters (pore size: 3 kDa, Merck Millipore Ltd. Tullagreen, Carrigtwohill, Co Cork, Ireland).
siRNAs
siRNAs used in this study were obtained from Nippon Gene Material Corp. (Tokyo, Japan). The siRNAs comprised the following sequences: USP9X #1, 5’-AGACUGGUUUCCACUUUUA-3’; USP9X #2, 5’-GAUGAGGAACCUGCAUUUC-3’; IRS-2 #1, 5’-UCGGCUUCGUGAAGCUCAAdTdT-3’; IRS-2 #2, 5’-GGCUGAGCCUCAUGGAGCAdTdT-3’; IGF-IR #1, 5’-GGAGUUCAAUUGUCACCAUdTdT-3’; IGF-IR #2, 5’-GGAUUGAGUUUCUCAACGAdTdT -3’; MMP9 #1, 5’-GGCCAAUCCUACUCCGCCU-3’; MMP9 #2, 5’-GGAGCCAGUUUGCCGGAUA-3’.
Transfection with plasmids or siRNA
The expression plasmids were transfected into PC3 cells using polyethylenimine (PEI) as described previously36. The plasmids were transfected into HEK293 cells using Lipofectamine 2000 (Life Technologies, Tokyo, Japan) via the forward transfection method following the manufacturer’s protocol. The siRNAs were transfected into PC3 cells using Lipofectamine RNAiMAX (Life Technologies, Tokyo, Japan) via the reverse transfection method, following the manufacturer’s protocol.
Gelatin zymography assay
Gelatin zymography was performed as follows: gels (SDS-PAGE, 7.5%) were copolymerized with gelatin (0.1%). Electrophoresis was performed using a mini gel slab apparatus (ATTO) with a constant voltage of 150 V at 4 °C until the dye reached the bottom of the gel. Following electrophoresis, gels were washed in wash buffer [Tris-HCl (50 mM, pH 7.5), Triton X-100 (2.5 %), CaCl2 (5 mM), ZnCl2 (1mM)] for 1 h. Then the zymograms were incubated overnight at 37 ℃ in incubation buffer [Tris-HCl (50 mM, pH 7.5), Triton X-100 (1 %), CaCl2 (5 mM), ZnCl2 (1mM)]. Gels were then stained with Coomassie brilliant blue (CBB) and destained with 49% methanol and 1% acetic acid. Areas of enzymatic activity appeared as clear bands on the blue background.
DNA synthesis assay
Cells were serum-starved for 48 h and then incubated with or without recombinant human IGF-I (100 ng/ml) for 20 h. DNA synthesis was measured using [³H] thymidine as described previously38.
Wound assay
PC3 cells transfected with siRNA were seeded into 12-well plates at 200,000 cells/well and incubated for 24 h. Then, a sterile 200-μl plastic pipette tip was used to create a scratch wound across the PC3 monolayer, following which the detached cells were removed by washing with cold phosphate-buffered saline [PBS; 137 mM NaCl, 2.68 mM KCl, 8.10 mM Na2HPO4 and 1.47 mM KH2PO4 (pH 7.4)] three times. Cells were then cultured in serum-free DMEM for 24 h and photographed at 0 and 24 h after scraping.
Real time PCR
Total RNA was extracted from cells using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA), and cDNA synthesis was performed using ReverTra Ace qPCR RT Master Mix (Toyobo, Osaka, Japan). cDNA was subjected to qPCR using THUNDERBIRD SYBR qPCR Mix (Toyobo) and Real-Time PCR system 7500TH (Life Technologies, Carlsbad, CA, USA). For all samples, 18S ribosomal RNA was used as an internal control for data normalization. The primers comprised the following sequences: human MMP9 forward, 5’-TACTCGACCTGTACCAGCGAG-3’; human MMP9 reverse, 5’-GGAATGATCTAAGCCCAGCGC-3’.
Immunostaining analysis
Cells were serum-starved for 20–24 h in DMEM supplemented with 0.1% BSA, and then incubated with or without human recombinant IGF-I (100 ng/ml) for 5 min. Cell lysates were prepared with lysis buffer (50 mM Tris-HCl, pH 7.4, 1 mM EDTA, 1 mM EGTA, 150 mM NaCl, 50 mM NaF, 1% Toriton X-100, 100 kallikrein-inactivating units/ml aprotinin, 20 mg/ml phenylmethanesulfonyl fluoride, 10 mg/ml leupeptin, 5 mg/ml pepstatin) and subjected to immunoprecipitation and immunoblotting as described previously36. The levels of each protein were quantified using ImageJ.
Prostate tissue study
Clinical prostate samples were collected for the Prostate: Evidence for Exercise and Nutrition Trial (PrEvENT) at Southmead Hospital, Bristol, with full ethical approval (Ethics 14/SW/0056)39. Male patients consented to allow prostate tissue samples to be obtained from the prostate that was removed at the time of their surgeries. All samples were stored in accordance with the Human Tissue Act 2004 and the study was performed in accordance with the Declaration of Helsinki. The sections examined were surpluses of the diagnostic requirements. Patient clinical and pathological data including age at surgery, pre-surgery PSA level, Gleason and histological grade, and lymph node invasion were extracted and analyzed anonymously from medical records for the purpose of this study.
The prostate tissues (n=100) were formalin-fixed and paraffin-embedded and 4 μm thick sections were cut using a microtome (Leica) and collected on Tomo adhesive microscope slides (Matsunami, Bellingham, WA, USA). Immunohistochemistry was performed using a Ventana BenchMark ULTRA™ machine (Roche, Oro Valley, AZ, USA) according to the manufacturer’s protocol. Briefly, sections were deparaffinized, pretreated with a cell conditioning step, and then incubated with the primary antibody against IRS-2 (Thermo Fisher, 1:5000). Sections were then counterstained with hematoxylin, dehydrated, and mounted in Clearium mounting medium (Leica). Slides were scored by two pathologists using a modified version of the Allred system, combining the proportion of tissue stained (on a scale of 1–5) with the staining intensity (on a scale of 1–3) to give a score out of 840.
Statistical Analysis
The results are presented as the mean ± standard error of the mean (SEM). Data were analyzed using one-way factorial ANOVA and Tukey–Kramer post-hoc multiple comparison tests. P < 0.05 was considered statistically significant (shown as “*” in graphs).