The following antibodies and reagents were used: antibodies against Hsp-90 #61041 (Becton Dickinson); PTEN #9559 (WB), pAkt (Ser473) #9271 (WB), #3787 (IHC), Akt #9272, pS6 #2211 (Cell Signaling); DICER #CG006 (Clonegene); MSCV-neo (Clontech); siGENOME non-targeting small interfering RNA (siRNA) #2 (si-Luc), si-miR-19a, si-miR-19b, si-miR-22, si-miR-25, si-miR-92a, si-miR-17, si-miR-20a, si-miR-93, si-miR-106b, si-miR-302a, si-miR-372, si-miR-373, si-PTEN, siGLO RISC-free control siRNA miRNA antisense inhibitor negative control #1, miR-19a antisense inhibitor, miR-22 antisense inhibitor, miR-25 antisense inhibitor, miR-93 antisense inhibitor, miR-106b antisense inhibitor, Dharmafect 1 (Dharmacon); miR-17, miR-19a, miR-20a, miR-22, miR-25, miR-93, and miR-106b 3′ digoxigenin (DIG)–labeled LNA ISH probes (Exiqon); lipofectamine 2000, Trizol reagent, deoxyribonuclease I (DNase I) amplification grade, SuperScript II reverse transcriptase, Dulbecco's modified Eagle's medium (DMEM), RPMI 1640, fetal bovine serum (FBS), keratinocyte serum-free medium, epidermal growth factor (EGF), bovine pituitary extract (BPE), antibody against Xpress Tag, pcDNA3.1/His C, geneticin (G418), hygromycin, antibody against PTEN (IHC) #51-2400 (Invitrogen); human MCM7 complementary DNA (cDNA) #MHS1011-75176 (Open Biosystems); pGL3-Control (Firefly luciferase), pRL-TK (Renilla luciferase), Dual-Luciferase reporter assay (Promega); fraction V bovine serum albumin (BSA), polybrene, insulin, antibody against actin #A3853, anti-FLAG antibody #F3165, puromycin (Sigma); QuantiTect Sybr Green PCRkit (Qiagen); antibody against MCM7 #9966 (Santa Cruz Biotechnology); QuikChange II XL Site-Directed Mutagenesis Kit, Herculase Taq polymerase (Stratagene); Ki67 antibody #VP-K451 (Vector Laboratories).
To construct the p1 and p2 chimeric luciferase plasmids, we removed the multicloning site of pGL3-control plasmid from its original position and inserted it into the Xba I site located downstream of the luciferase STOP codon. In this way, pGLU plasmid was obtained. An ~1.7- (p1) or 0.5-kilo–base pair (kbp) (p2) fragment of PTEN
3′UTR was then amplified from genomic DNA and cloned into the Kpn I and Xho I sites of pGLU. The QuikChange II XL Site-Directed Mutagenesis Kit was used to generate the mutated forms of these plasmids (fig. S1C
PIG/22 retroviral vector was obtained by cloning a ~0.55 genomic fragment encompassing human pri-miR-22
into Bgl II and Xho I sites of MSCV-PIG (51
) vector (puromycin resistance). The ~0.8–kbp-long intron 13 of human MCM7
was cloned analogously and PIG/106b~25 vector was obtained. The corresponding MSCV plasmids (hygromycin resistance) were obtained by subcloning in Bgl II and Xho I sites.
MSCV-neo-Flag-PTEN was generated by cloning the open reading frame of human PTEN (NM_000314.4) in the Eco RI and Xho I sites of MSCV-neo. The Flag epitope (AspTyrLysAspAspAspAspLys) was then inserted upstream of the ATG in Eco RI.
pSUPER-shEGFP and pSUPER-sh-Pten plasmids were previously generated in the laboratory (32
). sh-Pten is complementary to nucleotides 274 to 291 in the open reading frame of both mouse and human PTEN.
Cells and culture conditions
Wild-type MEFs were isolated from 13.5-day mouse embryos. After the legs, tail, liver, and head were removed, each embryo was mechanically fragmented and then incubated with trypsin–phosphate-buffered saline (PBS) 1:3 (10 ml total) at 37°C for 5 min. Five milliliters was removed and collected in 20 ml of DMEM + 10% FBS. Fresh trypsin (5 ml) was then added and the above-mentioned procedure was repeated three to four times. The collected cells were then spun down and seeded in a 6-cm dish. Cells were trypsinized at confluency (passage 1, P1). The propagation protocol 3T9 (9 × 105 cells per 100–mm-diameter dish transferred every 3 days) was followed. MEFs at P1 to P3 were used.
U2OS, Phoenix A and E cells were grown in DMEM +10% FBS. RWPE-1, PWR-1E, and Ca-HpV-10 were grown in keratinocyte medium + EGF + BPE. 22Rv1, DU145, LnCap, MDA-PCa-2b, and PC3 were grown in RPMI 1640 + 10% FBS. All cell lines were obtained from American Type Culture Collection and grown in penicillin-streptomycin and glutamine-containing medium at 37°C in a humidified atmosphere with 6% CO2.
Dual-luciferase reporter assay
DU145 cells were seeded at a density of 6 × 104
cells per 24-well plate. Twenty-four hours later, 400 ng of p1 or p2 plasmid per well were cotransfected with 80 ng of pRL-TK. Lipofectamine 2000 was used as transfectant according to the manufacturer's recommendations. Twenty-four hours after transfection, luciferase activity was measured and normalized as in (20
). In the case of plasmid-miRNA inhibitor cotransfection, 200 ng of p1/p1-mut-17/302 and 10 nmol of I-C/I-93 were transfected. Cells were analyzed as described above.
DU145 (1.5 × 105) and PWR-1E (0.8 × 105) were seeded in 12-well dishes, respectively. The following day they were transfected with 100 nM siRNAs/si-miRNAs or 400 nM miRNA inhibitors with Dharmafect 1 according to the manufacturer's recommendations. With this protocol, more than 90% of cells were positive for the fluorescent siGLO RISC-free control siRNA. Six hours after transfection, cells were seeded for growth curves (see below). Otherwise, the day after transfection, cells were trypsinized and reseeded in 12-well plates for subsequent collection and analysis.
DU145 and MEF retroviral infection
For retrovirus-mediated gene transfer of DU145, Phoenix A cells (3 × 106) were plated in a 10-cm poly-d-lysine–coated dish and, 16 hours later, were transfected with PIG retroviral plasmids using Lipofectamine 2000. Forty-eight hours later, the virus-containing medium (10 ml) was filtered and mixed with 5 ml of freshly prepared medium supplemented with polybrene (4 μg/ml). Cells (5 × 105) were plated in a 10-cm dish. Sixteen hours later, the medium was replaced with viral supernatant. Puromycin (2 μg/ml) was administered 48 hours after infection. The cells were selected for 2 days and then used for the various assays. For double infection, Phoenix A cells were transfected with empty PIG or the miRNA-expressing plasmids (PIG/22, PIG/106b~25), which carry puromycin resistance, and MSCV-neo empty or MSCV-neo-Flag-PTEN plasmids, which carry G418 resistance. The medium for infection contained 5 ml of freshly prepared medium, 5 ml of puro-resistant viral supernatant, and 5 ml of G418-resistant viral supernatant. Selection with puromycin (2 μg/ml) + G418 (500 μg/ml) was started 48 hours after infection and continued for 1 week. The selection medium was changed daily.
Double infection of MEF was performed similarly, except that Phoenix E cells instead of A cells were used. Phoenix E cells were transfected with MCSV/22 or MCSV/106b~25 plasmids, which carry hygromycin resistance (51
), and PIG/c-MYC, PIG/RasV12
, PIG/E1A (51
), or PIG/MCM7 puromycin-resistant plasmids. For sh-Pten experiments, Phoenix E cells were transfected with pSUPER-shPten, which carries puromycin resistance, and MSCV/c-MYC, MSCV/RasV12
, MSCV/E1A, or MSCV/MCM7 hygromycin-resistant plasmids. The medium used to infect MEFs contained 5 ml of freshly prepared medium, 5 ml of hygromycin-resistant viral supernatant, and 5 ml of puromycin-resistant viral supernatant. Forty-eight hours after infection, selection was started and carried on for 4 days [the first two with puromycin (2 μg/ml) + hygromycin (75 μg/ml), the second two with only hygromycin (75 μg/ml)]. The selection medium was changed daily.
DU145 cells were seeded at 3 × 105
cells per six-well dish. The following day, they were transfected with the different si-miRNAs as described. Six hours after transfection, cells of one six-well plate were trypsinized and replated in two 10-cm plates. The following day, they were labeled for 24 hours with [3
H]inositol (10 mCi/ml) for 24 hours in inositol-free DMEM supplemented with 10% FBS and 0.5% BSA. They were then serum-starved for 24 hours in inositol-free DMEM with [3
H]inositol (10 mCi/ml) and 0.5% BSA, but without FBS. After 5 min of stimulation with 200 nM insulin, cells were lysed in 1 M HCl. Lipids were extracted in chloroform-methanol (1:1, vol/vol) and deacylated as described (52
). Phosphatidylinositides were separated by anion-exchange high-performance liquid chromatography (Beckman), detected by a flow scintillation analyzer (Perkin-Elmer), and quantified with ProFSA software (Perkin-Elmer). The 3
H-labeled PI3P (phosphatidylinositol 3-phosphate), PIP2
, and PIP3
peaks were identified by 32
P-labeled in vitro synthesized internal lipid standards, prepared with baculovirus-expressed PI3K. For the [3
H]inositol labeling, the counts in each peak were normalized against the counts found in the phosphatidylinositol peak.
Total RNA was extracted with Trizol reagent according to the manufacturer's instructions.
Mature miRNAs were detected with TaqMan miRNA assays (Applied Biosystems) at the Biopolymers Facility (Harvard Medical School). RNU24 (human) or sno202 (mouse) were used as internal standards.
To determine the length of PTEN 3′UTR, total RNA was subjected to DNase treatment and retrotranscription (1 μg of RNA per vial). PCR was then performed with Herculase Taq polymerase.
To evaluate hPTEN, mPten, hMCM7, and intron 13 mRNA concentrations, real-time PCR was carried out with Sybr Green fluorescence. Two microliters of cDNA was used in a 20-μl reaction. ACTIN (human) or GAPDH (mouse) was used as an internal standard.
Relative quantification of gene expression was performed with the comparative CT
Transfected cells grown for specified time points were collected and lysed [50 mM tris (pH 8.0), 1 mM EDTA, 1 mM MgCl2, 1% NP-40, 1 mM β-glycerophosphate, 1 mM Na3VO4, 1 mM NaF, and protease inhibitors]. Proteins (30 μg per lane) were separated on 10% SDS–polyacrylamide gel and transferred to nitrocellulose membranes. Immunoblotting of the membranes was performed with the following primary antibodies: antibodies against PTEN (1:1000), Flag (1:1000), pAkt (Ser473) (1:1000), Akt (1:1000), MCM7 (1:2000), XPress (1:1000), Actin (1:10000), and Hsp90 (1:1000). Signals were revealed after incubation with the recommended secondary antibody coupled to peroxidase by enhanced chemiluminescence. Scanned images were quantified with ImageJ software.
In situ hybridization
ISH on TMAs was performed on 5-μm paraffin sections with 3′ DIG– labeled miRNA LNA (locked nucleic acid) probes with an automatic stainer (Discovery XT, Ventana Medical Systems Inc.). Cells were baked overnight at 60°C, dewaxed, postfixed in 4% paraformaldehyde (PFA) for 12 min, and then digested in proteinase K solution (15 μg/ml) for 4 min. Hybridization was performed overnight at 22°C below the melting temperature (Tm) of each probe with a commercial hybridization buffer (RiboHybe, Ventana Medical Systems). Two 16-min posthybridization washes in 2× SCC were performed at 4°C above the hybridization temperature. Then, sections were incubated for 40 min with a biotinylated antibody against DIG (InnoGenex). Detection with streptavidin–alkaline phosphatase and BCIP/NBT (bromochloroindolyl phosphate–nitro blue tetrazolium) substrates was performed for 10 hours with the BlueMap kit (Ventana Medical Systems). A Nikon Eclipse 50i microscope was used.
IHC was performed on 5-mm paraffin sections with the avidin-biotin-peroxidase method. The following primary antibodies were used: Ki67 (1:1000), pAkt (Ser473) (1:30), PTEN (1:100), pS6 (1:500), and DICER (1:250). Antigen retrieval was performed with citrate buffer (Ki67, DICER, pS6, PTEN) or 1 mM EDTA solution (pAkt). pAkt staining was considered specific only when cytoplasmic, and tumor cases were considered positive when staining in neoplastic cells was stronger than in normal prostate epithelium. PTEN abundance was scored semi-quantitatively with an intensity × quantity product (intensity: 0 = negative, 1 = weak, 2 = moderate, 3 = strong; quantity: 0 = negative, 1 = 1 to 9% of examined cells positive, 2 = 0 to 39% of cells positive, 3 = 40 to 69% of cells positive, 4 = 70 to 100% of cells positive). Scores ranging from 0 to 12 were averaged across replicate cores. For DICER, a classic three-category–based score was used, as shown in . A Nikon Eclipse 50i microscope was used.
Six hours after transfection, the cells of one 12-well dish were trypsinized, resuspended in 50 ml of medium, and plated in eight sets of three wells of a 12-well plate. Starting from the following day (day 0), 1 set of wells per day was washed once with PBS, fixed in 10% formalin solution for 10 min at room temperature, and then kept in PBS at 4°C. At day 7, all the wells were stained with crystal violet. After lysis with 10% acetic acid, the absorbance was read at 595 nm.
Growth in semisolid medium
The bottom layer was obtained by covering six-well dishes with 3 ml of 0.6% agar in DMEM. The following day, 5 × 104 infected cells were plated on this bottom layer in triplicate in 2 ml of 0.3% agar in DMEM + 10% FBS. Colonies were counted after 3 to 4 (DU145) or 4 to 6 (MEF) weeks at 40× and 100× magnification, respectively. Five fields for each well were counted. A Nikon Eclipse TE300 microscope was used. Images were acquired with IPLab software.
Injection into nude mice
NCR nude mice strain (4 to 6 weeks old; Taconic) were subcutaneously injected into the right flank with 4 × 106 infected cells. Tumor size was assessed weekly by caliper measurement. Tumor volume was calculated with the formula D × d2 × π/6, where D and d are the long and the short sides of the tumor.
Patient material and TMAs
Four TMAs were constructed from archival formalin-fixed, paraffin-embedded (FFPE) radical prostatectomy specimens from 184 cases of prostate cancer from the Department of Pathology of Brigham and Women's Hospital. Representative regions of each tumor were selected for coring on the basis of the corresponding hematoxylin and eosin (H&E)–stained full section. Three 0.6-mm-diameter cores of each tumor were included in the TMAs along with three cores of normal prostate tissue from the same patient (for 177 cases). Three cores with foci of PIN were available for 24 cases. Informed written consent was obtained from all patients, and the use of patient material in this study was approved by our internal institutional review board. Clinicopathological characteristics of the cohort of patients included in the study are summarized in table S3
Generation of MCM7 and MCM7i13 transgenic mice
Inserts from pcDNA/MCM7 and pcDNA/MCM7i13 plasmids (fig. S8A
) were excised by Hind III/Eco RI digestion and ligated to the Pst I/Eco RI– restricted ARR2
PB-containing plasmid (33
), after blunting one extremity to obtain the Pb/MCM7 and the Pb/MCM7i13 plasmids. The fragments containing Pb-MCM7-SV40polyA and Pb-MCM7i13-SV40polyAwere released by Not I/Pvu I digestion and injected into the pronuclei of fertilized oocytes in a B6/CBA F1 hybrid background. Southern blot analysis was performed according to standard procedures. The PCR primers used for genotyping are the following: forward primer, 5′ GCTAGAACTAGTGGATCCCCC 3′, and reverse primer, 5′ CATCGTCGTACAGATCCCGAC 3′
Prostates were extracted from euthanized mice and fixed in 4% PFA overnight at 4°C. They were then transferred into 70% ethanol, embedded in paraffin, sectioned and stained with H&E in accordance with standard procedures. IHC and ISH on prostate sections were performed as described above.
In vitro and in vivo data were analyzed with unpaired t
test (GraphPad Prism, GraphPad Software Inc.). Values of P
< 0.05 were considered statistically significant (*P
< 0.05; **P
< 0.01; ***P
< 0.001). The mean ± SD of three independent experiments is reported. Chi-squared test was used for the analysis of TMA samples (http://www.quantpsy.org