Loss of Sirt1 Promotes Prostatic Intraepithelial Neoplasia ... - Core

The American Journal of Pathology, Vol. 185, No. 1, January 2015

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See related Commentary on page 26 TUMORIGENESIS AND NEOPLASTIC PROGRESSION

Loss of Sirt1 Promotes Prostatic Intraepithelial Neoplasia, Reduces Mitophagy, and Delays Park2 Translocation to Mitochondria Gabriele Di Sante,*y Timothy G. Pestell,*y Mathew C. Casimiro,*y Sara Bisetto,*y Michael J. Powell,*y Michael P. Lisanti,yz Carlos Cordon-Cardo,x Mireia Castillo-Martin,x Dennis M. Bonal,x Valentina Debattisti,{ Ke Chen,*y Liping Wang,*y Xiaohong He,k** Michael W. McBurney,k** and Richard G. Pestell*y From the Departments of Cancer Biology* and Stem Cell Biology and Regenerative Medicine,z Sidney Kimmel Cancer Center,y and the Department of Pathology, Anatomy, and Cell Biology,{ MitoCare Center, Thomas Jefferson University, Philadelphia, Pennsylvania; the Department of Pathology and Urology,x Herbert Irving Comprehensive Cancer Center, Columbia University, New York, New York; and the Departments of Medicine and Biochemistryk and Microbiology and Immunology,** Ottawa Health Research Institute, University of Ottawa, Ottawa, Ontario, Canada Accepted for publication September 24, 2014. Address correspondence to Richard G. Pestell, M.D., Ph.D., Department of Cancer Biology, The Kimmel Cancer Center, Thomas Jefferson University, 233 S 10th St, Suite 1050, Philadelphia, PA 19107. E-mail: director@ kimmelcancercenter.org.

Prostatic intraepithelial neoplasia is a precursor to prostate cancer. Herein, deletion of the NADþ-dependent histone deacetylase Sirt1 induced histological features of prostatic intraepithelial neoplasia at 7 months of age; these features were associated with increased cell proliferation and enhanced mitophagy. In human prostate cancer, lower Sirt1 expression in the luminal epithelium was associated with poor prognosis. Genetic deletion of Sirt1 increased mitochondrial superoxide dismutase 2 (Sod2) acetylation of lysine residue 68, thereby enhancing reactive oxygen species (ROS) production and reducing SOD2 activity. The PARK2 gene, which has several features of a tumor suppressor, encodes an E3 ubiquitin ligase that participates in removal of damaged mitochondria via mitophagy. Increased ROS in Sirt1/ cells enhanced the recruitment of Park2 to the mitochondria, inducing mitophagy. Sirt1 restoration inhibited PARK2 translocation and ROS production requiring the Sirt1 catalytic domain. Thus, the NADþ-dependent inhibition of SOD2 activity and ROS by SIRT1 provides a gatekeeper function to reduce PARK2-mediated mitophagy and aberrant cell survival. (Am J Pathol 2015, 185: 266e279; http://dx.doi.org/10.1016/j.ajpath.2014.09.014)

The sirtuin family is a highly conserved group of NADþdependent histone deacetylases.1,2 The SIRT1 human homologue of the yeast Saccharomyces cerevisiae, the Sir2 gene, regulates co-activators (p300/calcium-binding protein),3 transcription factors [androgen receptor (AR) and forkhead box O], and components of the cell cycle, including the retinoblastoma protein.4e6 The effect of SIRT1 on cell proliferation varies with cell type and physiological conditions. SIRT1 promotes cell proliferation in colon, breast, and lung cancer cells and in tissue culture.5,7,8 In contrast, SIRT1 inhibits prostate cancer (PCa) cell proliferation and tumor growth and inhibits expression and activity of the AR.9 In the prostate of Sirt1/ mice, cellular proliferation was enhanced, associated with altered expression of androgen-responsive genes and histological feature of prostatic intraepithelial neoplasia (PIN).10 Copyright ª 2015 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ajpath.2014.09.014

The onset and progression of human PCa involve a series of distinct molecular genetic events.11 An understanding of the molecular mechanisms governing PCa is important because PCa remains the second leading cause of cancer death in the United States, with approximately 190,000 new Supported in part by NIH grants R01CA70896, R01CA75503, and R01CA86072 (R.G.P.), NIH Cancer Center Core grant P30CA56036 (R.G.P. for work conducted at the Sidney Kimmel Cancer Center), the China Scholarship Council (C.W.), the Pennsylvania Department of Health grant (C.W. and R.G.P.), by the American-Italian Cancer Foundation Postdoctoral Research Fellowship (G.D.), and Thomas Jefferson University resources (M.P.L. and his laboratory). The Pennsylvania Department of Health specifically disclaims responsibility for any analyses, interpretations, or conclusions. Disclosures: None declared. Current address of M.P.L., School of Cancer and Enabling Sciences, Faculty of Medical and Human Science, The University of Manchester, Manchester, UK.

SIRT1 Governs SOD2, PARK2, and Mitophagy cases diagnosed and 27,000 deaths occurring annually.12 The morphological features of PCa progression involve changes from PIN to invasive adenocarcinoma and metastases.11 Deletion of Pten, forced expression of Myc, Nkx3-1, and loss of Akt1 in transgenic mice are associated with PIN.13,14 Histological markers of PIN include increased expression of TP63 and a-methylacyl-CoA racemase (AMACR). TP63, which encodes a homologue of the TP53 gene, is highly expressed in the basal prostate cell in normal glands and PIN. The AMACR gene encodes a luminal marker protein that is overexpressed in prostate carcinoma.15,16 The combination of TP63 and AMACR is useful for diagnosing PIN and early adenocarcinoma. Cellular proliferation in prostate epithelial cells is a dynamic equilibrium of proliferation, apoptosis, and macroautophagy. Macroautophagy is an evolutionarily conserved catabolic process, requiring the formation of autophagosomes that engulf macromolecules and organelles within a cell. Autophagy is dynamically regulated by nutrient deprivation and cellular stress, including reactive oxygen species (ROS), and is thought to play a role in diverse functions and diseases, including aging, neurodegeneration, and tumorigenesis.17e19 A prosurvival function of autophagy aids in the clearing of damaged proteins, aggregates, or organelles, and it provides a cell with energy and anabolic building blocks.20,21 Alternatively, autophagy may be a mechanism of caspase- and apoptosis-independent cell death.22,23 The role of autophagy in tumor onset and progression is complex, with evidence for both tumor suppression and therapeutic resistance, depending on the type and stage of the tumor.24,25 Pancreatic cancer requires autophagy for tumor growth.26 In contrast, several studies demonstrated that autophagy contributes to tumor cell killing by exogenous agents.27,28 Increased BECN1 abundance, an inducer of autophagy, correlates with autophagy, and BECN1 haploinsufficiency functions as a tumor-suppressor gene.25 Herein, we show direct evidence that Sirt1 inhibits PIN in vivo using Sirt1/ mice. We show Sirt1 inhibits ROS via deacetylation and activation of mitochondrial superoxide dismutase 2 (Sod2), thereby reducing Park2 recruitment and autophagy. We propose that SIRT1 functions as a tumor suppressor in the prostate by inhibiting ROS and maintaining the physiological status of the autophagy/mitophagy process.

Materials and Methods Transgenic Mice and Genotyping Sirt1 transgenic mice were provided by an author (M.W.M.) and have been previously described.10 The appropriate institutional committeeeapproved protocols were followed when working with these mice. To confirm the genotype of the mice, a PCR analysis was conducted on extracted tail DNA using an oligonucleotide pair directed toward exon 5 of Sirt1 and a single Pgk1 cassette primer specific for the knockout allele, generating a Sirt1 endogenous amplimer of 423 bp and a

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Sirt1-Pgk1 knockout vector amplimer of 526 bp [nucleotide sequences, 50 -TTCACATTGCATGTGTGTGG-30 (forward) and 50 -TAGCCTGCGTAGTGTTGGTG-30 (reverse); and Pgk1, 50 -ATTTGGTAGGGACCCAAAGG-30 (reverse)], respectively.10

Gross Anatomical Analysis and IHC Sirt1þ/þ and Sirt1/ mice, aged 3 and 7 months, were euthanized by CO2 asphyxiation. Animals were dissected, with the following organs being removed: ventrodorsolateral (VDL) prostate and anterior prostate (AP) for hematoxylin and eosin (H&E) and immunohistochemical (IHC) staining. Portions of each organ were fixed in 4% paraformaldehyde to be used for generating sections. H&E staining was conducted along with IHC staining. The following were used: AMACR (catalog number 107916; Santa Cruz Biotechnology, Dallas, TX), Ki-67 (catalog number M7240; Dako, Carpinteria, CA), BECN1 (catalog number 11427; Santa Cruz Biotechnology), Nip3-like protein X (BNIP3L; catalog number ab8399; Abcam, Cambridge, MA), light chain (LC)-3 (symbol Map1lc3a; catalog number 4108S; Cell Signaling, Danvers, MA), SOD2-AcK68 [provided by Dr. David Gius29 (Northwestern University, Chicago, IL)], and phospho-50 -adenosine monophosphate-activated protein kinase (pAMPK) (catalog number 2531S; Cell Signaling).

Paraffin Embedding and Tissue Sectioning Paraformaldehyde-fixed tissues were subjected to an overnight (O/N) incubation in 70% ethanol, followed by a 4-hour incubation in 95% ethanol and a final O/N incubation in 100% ethanol. After the ethanol treatments, tissues were incubated in Histo-Clear (catalog number 38-7042; Ward’s Natural Science, Rochester, NY) for 4 hours at room temperature. The samples were exposed to a 1:1 ratio of wax (catalog number 470045-564; Ward’s Natural Science) and Histo-Clear for 4 hours, followed by a final, 4-hour wax incubation. After this, paraffin-embedded tissues were divided into sections using a microtome and mounted onto glass slides.

Immunofluorescence Paraffin-embedded slides of Sirt1þ/þ and Sirt1/ VDL prostates were incubated first at 60 C for 15 minutes. Tissues were then deparaffinized using the following solution gradient: xylene, 3 for 3 minutes; 100% ethanol, 3 for 3 minutes; 95% ethanol, 3 for 3 minutes; 70% ethanol, 3 for 3 minutes; and sterile double-distilled water (ddH2O), 1 for 5 minutes. Slides were microwaved in 1 Citra Antigen Retrieval Solution (catalog number HK086-9K; BioGenex, Fremont, CA) for 15 minutes and allowed to cool at room temperature. After two phosphate-buffered saline (PBS) washes (all washes throughout were 5 minutes), samples were incubated in Dako peroxidase blocking solution (catalog number 003715; Dako) at room temperature for 10 minutes, followed by two PBS with Tween 20 washes and one PBS

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Di Sante et al wash. Next, samples were blocked in normal goat serum (catalog number S-1000; Vector Laboratories, Burlingame, CA) at room temperature for 30 minutes and washed. Samples were then incubated for 1 hour in a working solution of mouse on mouse (M.O.M.), a mouse IgG blocking reagent at room temperature (catalog number BMK-2202; Vector Laboratories). After washing, tissues were incubated in a working solution of M.O.M. diluent for 5 minutes and incubated in primary antibody, anti-TP63 (catalog number 8431; Santa Cruz Biotechnology), reconstituted in M.O.M. diluent O/N at 4 C. After washes, tissues were incubated at room temperature for 10 minutes in M.O.M. biotinylated anti-mouse IgG reagent and subsequently washed. Next, sections were incubated in secondary antibody (catalog number A11029, Alexa Fluor 488; Invitrogen Molecular Probes, Grand Island, NY) at room temperature for 30 minutes, followed by a final wash. Sections were subsequently mounted in prolong gold antifade reagent with DAPI (catalog number P36931; Invitrogen Molecular Probes), coverslipped, and analyzed by confocal microscopy (Nikon Cl PLUS; Nikon, Melville, NY).

Cell Culture Sirt1þ/þ and Sirt1/ 3T3 cells were maintained in regular culture media (Dulbecco’s modified Eagle’s medium) supplemented with 10% FBS, 2 mmol/L L glutamine, 100 U/ mL penicillin, and 100 mg/mL streptomycin in humidified atmosphere containing 5% CO2 at 37 C.

Electroporation Sirt1þ/þ and Sirt1/ 3T3 cells (1 106) were transfected by electroporation (Nucleofector 2b Device, Program NIH 3T3 U-030; Lonza, Allendale, NJ) with the expression vectors pDsRed2-MITO (catalog number 632421; Clontech, Mountain View, CA) and 23955 YFP-PARK2 (catalog number 23955; Addgene, Cambridge, MA). Analysis was conducted by confocal (Nikon Cl PLUS) and live (Zeiss Axiovert Zoom; Zeiss, Dublin, CA) microscopy.

Live Imaging The Sirt1þ/þ and Sirt1/ 3T3 cells were electroporated with expression plasmid DNA and seeded onto 6-cm tissue culture plates for live imaging (P35G-1.5-14C; MatTek Corporation, Ashland, MA). The cells were allowed to recover for 12 hours in cell culture media, after which the regular medium was replaced with Dulbecco’s modified Eagle’s medium phenol-free medium supplemented with 10% FBS, 2 mmol/L L glutamine, 100 U/mL penicillin, and 100 mg/mL streptomycin. The cells were analyzed by live microscopy for 4 hours in the presence of 1 mmol/L carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP; catalog number C2920; Sigma-Aldrich, St. Louis, MO), an uncoupler of mitochondrial oxidative phosphorylation. The recruitment of Park2 was quantitated by calculating the area

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of each Park2 aggregate within the cell using ImageJ software version 1.48a (NIH, Bethesda, MD).

ROS Detection Cellular ROS was measured by a fluorogenic probe (catalog number C10422; Invitrogen Molecular Probes). The cellpermeable dye is non-fluorescent while in a reduced state, and exhibits bright fluorescence on oxidation by ROS. H2DCFDA at 5 mmol/L was used to stain the cells for 30 minutes, followed by fluorescence-activated cell sorting of stained cells at excitation and emission wavelengths of 492 to 495 and 517 to 527 mm, respectively. H2DCFDA fluorescence is induced on removal of the acetate group on oxidationinduced activation of intracellular esterases. Cellular fluorescent intensity was analyzed by fluorescence-activated cell sorting. The quantitated fluorescent signal was compared between the two cell lines and in the presence of ROS inhibitors, as previously described30: 5 mmol/L N-acetyl-L-cysteine (NAC; catalog number A9165; Sigma-Aldrich), 10 mmol/L ammonium pyrrolidinedithiocarbamate (PDTC; P8765; Sigma-Aldrich), and 5 mmol/L rotenone (catalog number R8875; Sigma-Aldrich). Hydrogen peroxide (50 mmol/L; catalog number P170-500; Fisher Scientific, Pittsburgh, PA) was used to enhance ROS production in 3T3 Sirt1þ/þ cells.30

Western Blot Analysis Whole-cell lysates (50 mg) were separated by 10% SDS-PAGE gel, and the proteins were transferred to nitrocellulose membrane for Western blot analysis, as previously described,31 and probed for SIRT1 (catalog number 15404; Santa Cruz Biotechnology), SOD2-AcK68 (provided by Dr. David Gius29), and b-tubulin (catalog number T4026; Sigma-Aldrich).

SOD2 Activity Assay All assays were performed in triplicate, according to Beauchamp and Fridovich.32 According to this assay, the proportion of active enzyme is directly proportional to the intensity of the bands. Sirt1þ/þ and Sirt1/ 3T3 cells (1.2 106) were plated in a 10-cm plate for 36 hours before protein lysis. Cells were washed three times with PBS not containing Ca2þ or Mg2þ. Then, 200 mL of native lysis buffer (2.7 mmol/L KCl, 1.5 mmol/ L H2PO4, 8 mmol/L NaHPO4, and 136.9 mmol/L NaCl, pH 7.0) was applied to the plate. Cells were harvested and sonicated using a Diagenode Bioruptor (Diagenode, Denville, NJ) on the high setting for 5 minutes, divided into 30-second on/off intervals. Lysates were then centrifuged using a tabletop centrifuge at 12,000 g for 5 minutes at 4 C. The supernatant was then transferred to a new collection 1.5-mL tube. Proteins were quantified using the BioRad Protein Assay, according to their specifications (catalog number 500-0006; Bio-Rad, Hercules, CA), and electrophoresed on a 12% stacking gel. Once 12% native gel was polymerized, it was run in preelectrophoresis buffer (for 1 L; 22.76 g of Tris and 0.38 g of

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SIRT1 Governs SOD2, PARK2, and Mitophagy

Figure 1 Sirt1/ mice show prostatic intraepithelial neoplasia (PIN) in prostate gland at 7 months. A: H&E and IHC staining of both anterior (AP) and ventro-dorsolateral (VDL) prostates in Sirt1þ/þ and Sirt1/ mice. H&E staining demonstrates presence of a focal atypical intraductal proliferation in Sirt1/ mouse prostate, compatible with PIN. The boxes represent areas considered for digital zoom magnification. a-Methylacyl-CoA racemase (AMACR) and Ki-67 immunostaining of both AP and VDL prostate in Sirt1þ/þ and Sirt1/ mice. BeE: Quantitation of AMACR immunoreactivity in Sirt1þ/þ and Sirt1/ AP (P Z 0.012) and VDL prostate (P Z 0.045). Ki-67eimmunoreactive cells in Sirt1þ/þ and Sirt1/ AP (P Z 0.004) and VDL (P Z 0.016) prostate. A Student’s t-test was performed on all comparisons. Whole data are shown as means SEM. n Z 3 separate mice. *P < 0.05, **P < 0.01.

disodium EDTA in 980 mL of ddH2O, pH to 8.8 and bring up to 1 L). Protein lysates (250 mg) were loaded in each lane, then run at 100 V for approximately 1 hour or until the samples were completely inside the stacking gel. Once they had entered the stacking gel, the pre-electrophoresis buffer was discarded and


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fresh/cold electrophoresis buffer (for 1 L; 6.06 g of Tris, 22.50 g of glycine, and 0.68 g of disodium EDTA) was added to 960 mL of ddH2O, pH to 8.3, and brought up to 1 L. The gel was then run at 100 V until the dye had reached the end of the gel. Once it had reached the end of the gel, it was run for another hour.

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Sirt1/ mice show a switch in the autophagy from 3- to 7-month-old prostates. A and B: Becn1 immunostaining of anterior prostate (AP) in Sirt1þ/þ and Sirt1/ mice aged 3 months and quantitation of immunoreactivity (P Z 0.0075). C and D: Becn1 immunostaining of AP in Sirt1þ/þ and Sirt1/ mice aged 7 months and quantitation of immunoreactivity (P Z 0.008). EeH: IHC staining for Becn1 in the stromal fibroblasts (red arrows) and epithelial cells (black arrows) in 7-month-old Sirt1þ/þ and Sirt1/ AP and ventrodorsolateral (VDL) prostates. Quantitation of Becn1 immunoreactivity in AP (P Z 0.023) and VDL (P Z 0.036) prostates. A Student’s t-test was performed on all comparisons. Whole data are shown as means SEM (n Z 3 separate mice). *P < 0.05, **P < 0.01.

Figure 2

The gel was removed from the electrophoresis apparatus and placed into a plastic container. The SOD2 stain (25 mg of Nitro Blue tetrazolium, 85 mL of tetramethylethylenediamine, 10 mg riboflavin-50 -phosphate, and 0.7 mg NaCN were dissolved in 50 mL of ddH2O) was applied to the gel and set to shake at room temperature in the dark for 20 minutes. After shaking, ddH2O was used to wash the gel twice; then, the gel was placed under a fluorescent light until white bands could be seen against the blue/ purple background. By using a Perfection V750 PRO scanner (Epson, Long Beach, CA), pictures were taken of the stained gel. Analysis of the scanned gels was performed using ImageJ.

Tissue Oxidative Stress Detection Tissue oxidative stress was detected using OxyIHC Oxidative Stress Detection Kit (catalog number S7450; Millipore, Billerica, MA). Paraffin-embedded prostate slides were deparaffinized and rehydrated [xylene, 2 minutes; xylene 1:1 with 100% ethanol, 3 minutes; 100% ethanol, 2 minutes; 95% ethanol,

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3 minutes; 70% ethanol, 3 minutes; 50% ethanol, 3 minutes; Ultra Pure H2O (catalog number D4641; Thermo Fisher Scientific, Waltham, MA)]. Antigen unmasking was performed adding 1 antigen retrieval buffer (20 minutes at 100 C). After three washes using 1 wash buffer (5 minutes each), dinitrophenylhydrazine solution was added (30 minutes at room temperature). After dinitrophenylhydrazine incubation, three washes were applied with 1 wash buffer (5 minutes each), and the unspecific antigens were covered with 1 blocking buffer (20 minutes at room temperature). The primary antibody was incubated on the slides in a humidified chamber O/N at 4 C. The slides were washed with 1 wash buffer (3 for 5 minutes) and incubated with a biotinylated secondary antibody (30 minutes at room temperature). After three washes using 1 wash buffer (5 minutes each), quenching was performed by incubating the slides with 3% H2O2 for 10 minutes at room temperature. The 3% H2O2 solution was removed with three 1 wash buffer washes (5 minutes each), and the slides were incubated with streptavidin-conjugated horseradish peroxidase

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Figure 3 Induction of mitophagy at 7 months in prostate of Sirt1/ mice. A: IHC staining of Nip3-like protein X (Bnip3l) and light cycle (LC)-3 in anterior (AP) and ventro-dorsolateral (VDL) prostates for Sirt1þ/þ and Sirt1/ mice. B and C: Quantitation of Bnip3l immunoreactivity in Sirt1þ/þ and Sirt1/ AP (P < 0.001) and VDL (P Z 0.013) prostate gland. D and E: Quantitation of LC-3 immunoreactivity in Sirt1þ/þ and Sirt1/ AP (P Z 0.016) and VDL (P Z 0.017) prostate. F and H: High magnification of the Bnip3l IHC staining in AP and VDL prostates for 7-month-old Sirt1þ/þ and Sirt1/ mice (red arrows, fibroblasts; black arrows, epithelial cells). G and I: Quantitation of Bnip3l immunoreactivity in the stromal fibroblasts and epithelial cells of Sirt1þ/þ and Sirt1/ AP and VDL prostate (P Z 0.040). A Student’s t-test was performed on all comparisons. Whole data are shown as means SEM. n Z 3 separate mice. *P < 0.05, ***P < 0.001.


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Induction of Trp63 and phospho-50 adenosine monophosphate-activated protein kinase (pAMPK) in Sirt1/ prostatic intraepithelial neoplasia (PIN) lesions. A: Trp63 (green) immunostaining of Sirt1þ/þ and Sirt1/ mouse ventrodorsolateral (VDL) prostates. The nuclei were stained with DAPI (blue). B: Quantitation of Trp63 immunoreactivity in Sirt1þ/þ and Sirt1/ VDL prostate (P < 0.001). C: pAMPK immunostaining of anterior prostate (AP) in 7-month-old Sirt1þ/þ and Sirt1/ mice. D: Quantitation of pAMPK immunoreactivity in 7-month-old Sirt1þ/þ and Sirt1/ AP (P Z 0.020). E: pAMPK immunostaining of VDL prostate in 7-month-old Sirt1þ/þ and Sirt1/ mice. F: Quantitation of pAMPK immunoreactivity in 7-month-old Sirt1þ/þ and Sirt1/ VDL prostate (P Z 0.002). A Student’s t-test was performed on all comparisons. Whole data are shown as means SEM. n Z 3 separate mice. *P < 0.05, **P < 0.01, and ***P < 0.001.

Figure 4

(30 minutes at room temperature). After the washes, the slides were incubated with 3,30 -diaminobenzidene [DAB A (catalog number CS205113, Millipore) and DAB B (catalog number CS205109, Millipore)] mixture solution and Ultra Pure H2O (Thermo Fischer Scientific) was used to stop the reaction. After the 3,30 -diaminobenzidene reaction, the slides were dehydrated (ethanol 100%, 2 for 5 minutes) and washed with xylene (3 for 5 minutes). Mounting medium was added to the samples, and the slides were covered with a plastic slip. The slides were allowed to dry O/N and visualized on a light microscope (Nikon Eclipse 50i).

Statistical Analysis

patient samples were examined. The relative expression of SIRT1 was reduced in patients with poor prognosis (Supplemental Figure S1A) (82% versus 67%; P Z 0.012). To determine the subcellular compartmentalization and cell type in which SIRT1 was expressed, human PCa and normal prostate tissue were double stained with SIRT1 and either cytokeratin 5 (KRT5; a basal epithelial cell marker) (Supplemental Figure S1B) or cytokeratin 8 (KRT8; a luminal epithelial cell marker) (Supplemental Figure S1C). KRT5-positive cells showed an increased abundance of nuclear SIRT1 in PCa tissues (P < 0.001) (Supplemental Figure S1D). KRT8-positive cells showed an increased abundance of cytoplasmic SIRT1 in PCa tissues (P Z 0.019) (Supplemental Figure S1E).

The statistical analysis was performed using Prism version 4 (GraphPad Software, San Diego, CA). P < 0.05 was considered statistically significant.

Sirt1/ Mice Develop PIN and Show an Increased AMACR and Ki-67 Staining

Results Reduced SIRT1 Expression in Poor Prognosis PCa To determine the predictive value and the biological significance of altered SIRT1 expression in human PCa samples, 154

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To determine the role of Sirt1 in the development of murine prostate PIN and autophagy, Sirt1þ/þ and Sirt1/ mice were examined at 7 months. On the basis of H&E staining, Sirt1/ mice showed morphological changes characteristic of PIN, including a larger stromal layer with increased cellularity and nuclear atypia in both AP and VDL prostate (Figure 1A and

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Figure 5 Seven-month-old prostate tissues and 3T3 Sirt1/ cells show an increase of endogenous reactive oxygen species (ROS) production. A: IHC staining for oxidized proteins in 3- and 7-month-old Sirt1þ/þ and Sirt1/ prostates. BeE: Quantitation of oxidized protein staining for 3-month-old mice anterior prostate (AP), ventro-dorsolateral (VDL), 7-month-old mice AP, and VDL. F: Fluorescence intensity spectrum for endogenous ROS measurement in unstained (magenta), 3T3 Sirt1þ/þ (blue), and Sirt1/ (red) cells. Endogenous ROS production is higher in Sirt1/ 3T3 cells (P < 0.003). G: ROS production was measured in 3T3 Sirt1þ/þ cells in the presence of ROS inducer hydrogen peroxide (P Z 0.001). H: ROS production was measured in 3T3 Sirt1/ cells in the presence of ROS inhibitors: rotenone (Rot), NAC, and PDTC (P < 0.001, P Z 0.006, and P Z 0.010, respectively). A Student’s t-test was performed on all comparisons. Whole data are shown as means SEM. n Z 3 separate experiments. *P < 0.05, **P < 0.01, and ***P < 0.001.


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Di Sante et al Supplemental Figure S2). AMACR and Ki-67 expression is highly correlated with PIN.33 IHC staining for AMACR and Ki-67 (Figure 1A and Supplemental Figure S2) was conducted on both the AP and VDL prostate. The AMACR abundance was increased fourfold in AP (P Z 0.012) (Figure 1B) and approximately twofold in VDL prostate of Sirt1/ mice (P Z 0.045) (Figure 1C). The number of Ki67epositive cells was increased approximately threefold in the Sirt1/ AP (P Z 0.004) (Figure 1D) and approximately fivefold in the Sirt1/ VDL (P Z 0.016) (Figure 1E) prostate.

Sirt1 Switch to Inhibitor of Mitophagy in Prostate Tissues in Vivo and Sirt1/ PIN Lesions Are Associated with Stromal Fibroblast Mitophagy Because mitophagy can contribute to cellular stress resistance and thereby promote cellular growth,19,34 we considered the possibility that increased mitophagy may have contributed to the ongoing increased growth of the Sirt1/ prostate. To determine whether ongoing cellular stress in the prostate of Sirt1/ mice may have induced autophagy, we compared the prostate of 3- and 7-month-old Sirt1þ/þ versus Sirt1/ mice. IHC staining for Becn1 on the histological prostate sections of 3- and 7-month-old mice demonstrated an increase in Becn1 in the 7-month-old Sirt1/ prostates (Figure 2, AeD). The 3-month-old Sirt1þ/þ mice show Becn1 abundance increased in the AP compared with the Sirt1/ mice (Figure 2A and Supplemental Figure S3A), with a relative abundance increased approximately threefold (P Z 0.0075) (Figure 2B). The 7-month-old Sirt1/ mice show a Becn1 abundance higher in the AP (Figure 2C and Supplemental Figure S3A), by more than twofold (Figure 2D) (P Z 0.008). Tumor progression is associated with the induction of autophagy in the tumor stroma, providing micronutrients to the tumor epithelial cells.35 To determine whether mitophagy was occurring in the stroma associated with the PIN lesions, quantitative IHC was conducted. Becn1 immunoreactivity was increased threefold in the Sirt1/ stromal fibroblasts in the AP prostate (Figure 2, E and F, and Supplemental Figure S3B) and 10-fold in the VDL prostate (Figure 2, G and H, and Supplemental Figure S3B), associated with PIN. Becn1 IHC staining was also increased in the epithelial cells of the AP (Figure 2, E and F, and Supplemental Figure S3B) and VDL (Figure 2, G and H, and Supplemental Figure S3B). At 7 months, the Bnip3l and LC-3 abundance in both Sirt1/ AP and VDL prostate cells was increased compared with Sirt1þ/þ cells (Figure 3A and Supplemental Figure S4A). The relative abundance of both autophagic markers was increased in both Sirt1/ AP and VDL (P < 0.001, P Z 0.013, P Z 0.016, and P Z 0.017, respectively) (Figure 3, BeE). Only minor changes in Bnip3l abundance were observed in Sirt1/ fibroblasts (Figure 3, F and G, and Supplemental Figure S4B). Bnip3l abundance was increased in the VDL epithelial cells (P Z 0.040) (Figure 3, H and I, and

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Supplemental Figure S4B). The inducing of Becn1 immunoreactivity at 7 months, indicative of mitophagy, contrasts with the finding at 3 months, in which Becn1 IHC was reduced in the Sirt1/ prostate (Figure 2A and Supplemental Figure S3A).10 Bnip3l localizes to the mitochondria to participate in mitophagy. We determined the relative colocalization of Bnip3l in Sirt1þ/þ versus Sirt1/ 3T3 cells. Bnip3l showed enhanced mitochondrial colocalization in Sirt1/ 3T3 cells (Supplemental Figure S4C). Thus, Sirt1 inhibits expression of the mitophagy marker, Becn1, and inhibits Bnip3l recruitment to mitochondria.

Trp63 and pAMPK Are Overexpressed in Sirt1/ Mice Prostates The TP63 protein is a homologue of TP53, which is highly expressed in basal prostate cells.36 Immunofluorescence staining for Trp63 was conducted on the VDL prostate (Figure 4A). The Trp63 abundance in the Sirt1/ prostate cells was increased approximately fourfold compared with littermate controls (P < 0.001) (Figure 4B). Recent studies have demonstrated induction of pAMPK in PIN.37 IHC for pAMPK was increased in the Sirt1/ compared with Sirt1þ/þ AP (P Z 0.020) (Figure 4, C and D, and Supplemental Figure S5) and VDL (P Z 0.002) (Figure 4, E and F, and Supplemental Figure S5).

Sirt1 Inhibits ROS Production and Induces Sod2 Activity We determined the oxidative stress to Sirt1þ/þ and Sirt1/ prostate tissues using the OxyIHC Oxidative Stress Detection Kit. At 3 months of age, Sirt1þ/þ mice show a greater basal oxidation than Sirt1/ mice in both AP (P < 0.001) and VDL Sirt1þ/þ prostates (Figure 5, AeC, and Supplemental Figure S6). However, at 7 months of age, Sirt1/ mice showed a greater basal oxidative stress in both the AP (P < 0.001) and VDL (P Z 0.013). Thus, with age, there is a relatively greater increase in oxidative stress in Sirt1/ prostates (Figure 5, A, D, and E, and Supplemental Figure S6). We next determined ROS production in Sirt1þ/þ and Sirt1/ 3T3 cells using a fluorogenic dye that exhibits fluorescence on oxidation by ROS. ROS production was increased 2.5-fold in Sirt1/ 3T3 cells (P Z 0.003) (Figure 5F). Hydrogen peroxideeinduced ROS production approximately twofold in Sirt1þ/þ 3T3 cells (P Z 0.001) (Figure 5G). To confirm the validity of these assays, well-characterized ROS inhibitors were used. ROS production in Sirt1/ 3T3 cells was reduced by rotenone, NAC, or PDTC (P Z 0.001, P Z 0.006, and P Z 0.010, respectively) (Figure 5H). SOD2 serves as a key detoxifying enzyme of ROS production. We, therefore, conducted Sod2 activity assays on Sirt1/ versus Sirt1þ/þ fibroblasts (Figure 6A). Sod2 activity was reduced in Sirt1/ fibroblasts (P < 0.001) (Figure 6B). To determine whether the reduction in Sod2

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Sirt1/ cells show a reduced superoxide dismutase 2 (Sod2) activity. A: Sod2 activity detection in 3T3 Sirt1þ/þ and Sirt1/ cells in presence and absence of ethanol. B: Sod2 activity quantification on the basis of band intensity (P < 0.001). C: Western blot analysis probed for anti-SIRT1, antieSOD2AcK68, and b-tubulin (as loading control). D: Densitometric analysis of SOD2-AcK68 (P < 0.001). EeH: Oncomine cancer-profiling database analysis of SOD2 expression in PCa versus normal tissue. Using the Tomlins database, SOD2 is down-regulated in benign prostatic hyperplasia (P < 0.001, fold change Z 3.19), PIN (P < 0.001, fold change Z 4.24), and prostate adenocarcinoma (P Z 0.047, fold change Z 2.06). Using the Tomlins database, SIRT1 is down-regulated in benign prostatic hyperplasia (P Z 0.015, fold change Z 1.547). Using the Wallace database, SOD2 is down-regulated in prostate adenocarcinoma (P < 0.001, fold change Z 1.965). Using the Wallace database, SIRT1 is down-regulated in prostate adenocarcinoma (P Z 0.015, fold change Z 1.10). The black circles in EeH represent outliers. ***P < 0.001. BPH, benign prostatic hyperplasia; NPG, normal prostate gland; PC, prostate adenocarcinoma; and PIN, prostatic intraepithelial neoplasia.

Figure 6

activity in Sirt1/ cells was due to reduced Sod2 abundance, we determined Sod2 mRNA and protein and promoter regulation by Sirt1. Sod2 expression is induced by ROS and is repressed by endogenous apoptosis-inducing genes, including Jun,30 E2f1, and Myc.38 In contrast with the increased Sod2 activity in Sirt1þ/þ fibroblasts, Sod2


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mRNA, protein, and promoter activity were repressed by Sirt1 (data not shown). Sod2 activity and the ability to scavenge ROS are inhibited by acetylation.39 We, therefore, considered that SIRT1 may regulate SOD2 activity through post-transcriptional mechanisms. SOD2 is acetylated by histone acetyltransferase, p300, and P300/CBP-associated

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Di Sante et al factor. Sod2 acetylation Western blot analysis was conducted using antieSOD-AcK68 antibody. A substantial increase in Sod2 acetylation was observed in the Sirt1/ fibroblasts (P < 0.001) (Figure 6, C and D). By using Oncomine, a cancer-profiling database, we compared both SOD2 and SIRT1 expression in different stages of human PCa versus normal tissue reported in two databases: Tomlins and Wallace. By using the Tomlins database, SOD2 (Figure 6E) and SIRT1 (Figure 6F) were downregulated in benign prostatic hyperplasia (P < 0.001, fold change Z 3.19, and P Z 0.015, fold change Z 1.547, respectively), SOD2 was also down-regulated in PIN (P < 0.001, fold change Z 4.24) and prostate adenocarcinoma (P Z 0.047, fold change Z 2.06) (Figure 6E). By using the Wallace database, SOD2 (Figure 6G) and SIRT1 (Figure 6H) were down-regulated in prostate adenocarcinoma (P < 0.001, fold change Z 1.965, and P Z 0.039, fold change Z 1.10, respectively).

Sirt1 Inhibits Park2 Translocation to Mitochondria To determine whether Sirt1 inhibition of mitophagy may involve the inhibition of Park2 recruitment, mitochondria of 3T3 Sirt1þ/þ and Sirt1/ cells were monitored by live microscopy. FCCP induces transient mitochondrial depolarization and recruitment of Park2. FCCP administration (0 minutes) induced Park2 recruitment in 3T3 Sirt1þ/þ versus Sirt1/ cells (55 minutes) (Figure 7, A and B). Park2 recruitment kinetics were monitored for 15 minutes at an interval of 5 minutes (60, 65, and 70 minutes) (Figure 7C). Park2 aggregate areas were quantitated. At 70 minutes, the recruitment of Park2 was significantly increased in Sirt1/ cells (P Z 0.039) (Figure 7C). Together, these studies are consistent with a model in which SIRT1 deacetylation of SOD2 induces SOD2 activity and reduces ROS production, PARK2 recruitment, and autophagy. The induction of autophagy in the local stress of the PIN lesions in Sirt1/ mice may provide key micronutrients known to promote aberrant epithelial cell growth.

Discussion Herein, deletion of Sirt1 induced PIN in 7-month-old mice, as evidenced by histological features of increased cellularity and Figure 7 Time lapse of Park2 recruitment during the mitophagy process in Sirt1/ 3T3 cells. A: Park2 (green) translocation into the mitochondria (red) was monitored by live microscopy in Sirt1/ cells after 1 mmol/L FCCP administration. Starting from FCCP administration (0 minutes), Park2 and mitochondrial signals were detected every 5 minutes. Park2 recruitment started at 55 minutes. The maximum recruitment of Park2 occurs in a frame of time lasting 15 minutes. B: Magnification of Park2 and mitochondria colocalization (arrows) at 70 minutes after phenylhydrazone (FCCP) administration. C: Quantitation of Park2 recruitment in 3T3 Sirt1þ/þ (black circles) and Sirt1/ (black triangles) cells at 60, 65, and 70 minutes (P Z 0.039). A one-way analysis of variance was performed for the statistical analysis. Data are shown as means SEM. n Z 5. *P < 0.05.

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SIRT1 Governs SOD2, PARK2, and Mitophagy nuclear atypia, glandular hyperplasia, and increased staining for Ki-67, Trp63, and AMACR in Sirt1/ mice.10 First, the current findings extend prior studies that suggested that PIN occurs in 3-month-old Sirt1/ mice prostates on the basis of H&E staining10 through the use of AMACR and Trp63 staining. Second, these studies demonstrate that PIN persists at 7 months in Sirt1/ mice. Third, these studies demonstrate mitophagy in both the epithelial and fibroblast compartment of the prostate in Sirt1/ mice. Fourth, these studies demonstrate an increase in intracellular oxidative stress in the Sirt1/ prostate. The role of SIRT1 as either an oncogene or a tumor suppressor may be cell-type specific; however, it is still controversial. The following studies have shown that SIRT1 may have an oncogenic role in advanced PCa. Huffman et al40 have demonstrated that Sirt1/SIRT1 is overexpressed in the mouse model and advanced human PCa, and it was also found to promote PCa cell growth and survival.41 Yang et al42 have also demonstrated that the tumor-suppressor activity of forkhead box O 1 is suppressed by SIRT1-mediated deacetylation in PCa cells, and Wang et al43 have found that SIRT1 is a key downstream effector of nicotinamide phosphoribosyltransferase for oxidative stress in prostate cancer. An overexpression of nicotinamide phosphoribosyltransferase in PCa cells along with SIRT1 leads to oxidative stress resistance.43 Moreover, SIRT1 promotes both cell invasion in PCa cells enhancing matrix metalloproteinase-2, which has been shown to play an important role in cancer cell invasion,44 and induction of epithelialto-mesenchymal transition through the promotion of the transcription factor ZEB1.45 However, the current studies demonstrate reduced SIRT1 levels in patients with PCa are associated with reduced recurrence-free survival in human PCa. Consistent with the current studies in which SIRT1 inhibits PIN, SIRT1 suppressed intestinal tumorigenesis in colon cancer.46 In these prior studies, SIRT1 deacetylated b-catenin and promoted cytoplasmic localization of b-catenin.46 The reintroduction of SIRT1 into TP53 mutant cancer cells suppresses tumor formation, likely related to a role of SIRT1 in DNA damage repair.47 In PCa cells, SIRT1 was shown to inhibit AR activity and cellular proliferation.9 The ability of SIRT1 to inhibit PCa cell proliferation in tissue culture involved the catalytic domain and correlated with AR deacetylation. The inhibition of AR expression and activity by SIRT1 is likely mediated in part through deacetylation of the AR.9,48e50 The current studies demonstrated increased cytoplasmatic SIRT1 in luminal cell prostate. Herein, endogenous Sirt1 inhibited mitophagy in the prostate of mice. The key autophagy proteins BECN1 and, in yeast, autophagy-related protein 5 (Atg5) or Atg9p are localized in mitochondria.51e53 Atg5 and microtubule-associated protein LC-3 localize to punctae on mitochondria.54 The BNIP3L is an important regulator of mitophagy55,56 and is primarily integrated into the mitochondrial membrane, and BNIP3L overexpression leads to loss of membrane potential and cell death. BECN1, BNIP3L, and LC-3 are important for the removal of mitochondria during the autophagic response.56 The relative abundance of the


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mitophagy markers Bnip3l, Becn1, and LC-3 was increased in the Sirt1/ prostate epithelium. Careful analysis of autophagy markers in the epithelial cells and the stroma of the Sirt1/ PIN lesions evidenced both epithelial cells and stroma fibroblast mitophagy.57 Herein, Bnip3l showed enhanced mitochondria localization in Sirt1/ fibroblasts. Cells remove damaged mitochondria to prevent the accumulation of ROS. Mitochondrial autophagy or mitophagy is a process of mitochondrial quality control. ROS, including superoxide anions, are byproducts of oxidative phosphorylation and are eliminated by the scavenger enzyme, Sod2, which converts superoxide anions to hydrogen peroxide.38 The PARK2 protein is recruited in a ROS-dependent manner to mitochondria binding to PTEN-induced putative kinase 1 and inducing mitophagy of depolarized mitochondria.58 Mitophagy allows for the selective removal of dysfunctional mitochondria by autophagosomes. Mitophagy may occur in a PARK2dependent or PARK2-independent manner. The induction of stroma fibroblast mitophagy has been proposed as a mechanism promoting the turnover of proteins, and the PIN lesions may be supported through the production of micronutrients released from the mitophagic microenvironment. The current studies demonstrate a shift from the 3- to 7-month PIN lesions in which Sirt1 induced autophagy at 3 months and inhibited autophagy at 7 months. SIRT1 has been shown to both inhibit and induce autophagy.59 What might explain the shift toward increased mitophagy in PIN lesions from 3 to 7 months? One explanation may be the progressive increase in ROS production, as evidenced by tissue oxidative stress detection (Figure 5, AeE). Herein, Sirt1 inhibited ROS production and induced Sod2 activity. ROS are induced by oncogene stimuli (Ras and ErbB2), and the induction of ROS may be due to the progressive reduction in SOD2 activity due to altered SOD2 acetylation status. Because Sirt1 is cytoplasmic and Sod2 is mitochondrial, the induction of Sod2 activity and reduction in Sod2 acetylation at lysine 68 in the Sirt1þ/þ compared with Sirt1/ may have been indirect because of the increase in mitochondrial Sirt3 (data not shown). How might the induction of mitophagy in the Sirt1/ prostate contribute to PIN? The induction of autophagy has been shown to promote tumor progression in the context of Myc-dependent tumor growth.34 AMPK promotes cancer cell survival in the presence of stressors, whether bioenergetics, growth factor, or oncogene induced. pAMPK was induced in the prostate of the Sirt1/ mice, AMPK can function as a context-dependent oncogene,34,60 and the induction of pAMPK is observed in other mouse models of PIN. Thus, the pAMPK induction may have contributed to the PIN phenotype in Sirt1/ mice.

Acknowledgments R.G.P. and M.P.L. developed the idea; R.G.P., G.D., and M.C.C. developed the experimental design; R.G.P. and

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Di Sante et al G.D. wrote the manuscript; G.D. performed organ dissections, immunostaining for AMACR, Ki-67, Becn1, Bnip3l, LC-3, and ROS, live imaging, and all of the analysis; T.G.P. performed the Sod2 activity assay; K.C. performed the luciferase assay; L.W. performed pAMPK staining; M.J.P. performed Trp63 staining and Bnip3l staining in 3T3 cells. C.C.-C., M.C.-M., and D.M.B. performed H&E and IHC staining with the autophagy markers on the tissues; M.W.M. and X.H. generated the Sirt1/ mice; S.B. managed the mouse colony and analyzed the AQUA staining; and V.D. performed mitochondrial fractionation.

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Supplemental Data 19.

Supplemental material for this article can be found at http://dx.doi.org/10.1016/j.ajpath.2014.09.014.

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