Background
Prostate cancer (PC) is the most frequently occurring male malignancy worldwide. In 2015, more than 220,000 new cases and 27,000 PC-related deaths were reported in the USA [
1]. Treatment options include surgery, radiation therapy, and androgen deprivation therapy (ADT). Although most patients initially respond to ADT, they frequently develop recurrent castrate-resistant PC (CRPC) [
2] for which management options are limited to aggressive chemotherapy and palliative care.
The androgen receptor (AR) is the central transcription factor in PC biology and pathogenesis. After binding to androgens, the AR translocates to the nucleus, forms homodimers, and binds androgen response elements (AREs) in the promoters of target genes, altering their expression. In primary PC, the inhibition of the AR pathway by anti-androgens leads to dramatic tumor regression. In CRPC, however, tumors acquire resistance to ADT but remain dependent on AR through molecular alterations including AR amplification, mutations, splice variants, as well as overexpression of AR co-activators [
3‐
6]. Co-activators include chaperone proteins, members of the p160 family, DNA repair proteins, ubiquitin ligases, histone demethylases, and acetyltransferases,
inter alia [
7]. Lysine (K) acetyltransferases (KATs) such as p300 and TIP60 have been reported to acetylate and activate the AR in metastatic PC [
8‐
12].
We identified the first member of the INhibitor of Growth (ING) family of epigenetic regulators using PCR-mediated subtractive hybridization between normal and cancerous breast epithelial cells [
13], and ING2–5 were subsequently identified by sequence homology [
14‐
16]. ING proteins are stoichiometric members of histone/lysine acetyltransferase (KAT; ING3–5) or histone/lysine deacetylase (KDAC; ING1, ING2) complexes [
17]. They specifically recognize H3K4Me3 and recruit KAT or KDAC complexes to alter the chromatin structure [
18]. We noted that Yng2, the budding yeast homolog of ING3, is a member of the NuA4 KAT complex, and that deletion of Yng2 caused severe cell cycle and growth defects [
19]. Affinity purification followed by mass spectrometry showed that human ING3 is an essential and stoichiometric member of the TIP60 KAT complex that is analogous to NuA4, and its role in this complex is conserved from yeast to mammals [
20].
Early studies reported that, similar to ING1, ING3 functions as a type II tumor suppressor to regulate apoptosis and is downregulated in cancers such as melanoma and head and neck carcinoma [
15,
21,
22]. However, more recent examination of ING3 function in regulating cardiac hypertrophy indicated a positive growth effect through mTOR [
23], and using new, more reliable immunological reagents than were previously available, we found that ING3 is highly expressed in proliferating human tissues such as skin, small intestine, and bone marrow [
24].
In a recent screening study, it was noted that high ING3 levels correlate with worse prognosis in patients with erythroblast transformation-specific-related gene (ERG) negative PC, and that a ten-gene signature that correlates with patient survival in these cancers included ING3 [
25,
26]. However, the mechanism by which ING3 contributes to ERG-negative PC and its role in PC biology in general were not characterized. Here, we show that ING3 is an AR co-activator, which promotes TIP60-mediated AR acetylation and nuclear translocation, leading to PC cell proliferation and migration. In addition, we provide evidence that ING3 levels correlate with AR levels in PC patient samples, and show that higher ING3 levels serve as a biomarker predicting poorer prognosis in patients with low AR expression.
Methods
Cell culture, plasmids, and transfection
LNCaP, VCaP, PC3, DU145, and HEK293T cell lines were purchased from American Type Culture Collection (ATCC), and C4-2 cells were a gift from Dr. Martin Gleave. All lines were periodically checked for mycoplasma by PCR. LNCaP, C4-2, and PC3 were grown in Roswell Park Memorial Institute (RPMI) medium supplemented with 10% fetal bovine serum (FBS). DU145, VCaP, and HEK293T cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS. For androgen deprivation, cells were incubated with media supplemented with 5% charcoal stripped FBS (CSS) (Invitrogen) for 48 h. Mibolerone (MB) (Toronto Research Chemicals) was used as an androgen analog at concentrations of 1–10 nM. The pCMV-3myc-AR plasmid was a gift from Dr. Marja Nevalainen. The pCIN4-FLAG-HA-TIP60 was a gift from Dr. Wei Gu. HEK293T cells were transfected using TransIT 293 reagent (Mirus), and PC cells were transfected using Lipofectamine LTX (Invitrogen). Knockdown of ING3 by small interfering ING3 (siING3) was done as previously described [
24].
Lentiviral short hairpin RNA (shRNA) generation and infection
Three shRNA sequences against ING3 (shING3) derived from the RNA interference (RNAi) codex and a scrambled shRNA (shCtrl) with similar GC content were cloned in pINDUCER10, a doxycycline (Dox)-inducible lentiviral vector [
27]. C4-2 cells were infected with concentrated inducible lentivirus encoding either shCtrl or shING3. After the addition of Dox, cells were sorted keying on red fluorescent protein (RFP) expression (Additional file
1: Figure S1).
Immunoprecipitation (IP)
For IP, 1 × 10
7 cells were lysed at 4 °C using lysis buffer (50 mM Tris-HCL, 150 mM NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA), 1% Triton X-100) supplemented with protease inhibitors (cOmplete, Roche). Antibodies including anti-HA (Roche), anti-Myc (Sigma), anti-acetyl lysine (Santa Cruz Biotechnology), anti-ING3 (Kerafast [
24]), anti-AR (N-20, Santa Cruz Biotechnology), or anti-TIP60 (C-7, Santa Cruz Biotechnology) were crosslinked to beads (GE Healthcare) and used for IP.
In vitro acetylation assays
HEK293T cells were transfected with green fluorescent protein (GFP), ING3-HA, or AR-Myc and were lysed 24 h post-transfection. GFP- or ING3-transfected cell lysates were incubated with anti-HA, and AR-transfected cell lysates were incubated with either anti-Myc or normal rabbit IgG (Santa Cruz). For acetylation assays, IP samples were washed once in histone acetyltransferase (HAT) buffer (50 mM Tris-Cl pH 8.0, 10% glycerol, 0.1 mM EDTA, 10 mM butyric acid, 2 μM TSA). HA-IP samples were mixed with protein A beads or control rabbit IgG IP. The AR-IP sample was divided equally into two tubes and mixed with HA-IP samples from either GFP- or ING3-transfected cell lysates. Following addition of 1 mM acetyl coenzyme A (Lithium salt, Sigma), all tubes were incubated at 30 °C for 1 h with occasional shaking.
Luciferase reporter assays
We plated 5 × 104 HEK293T cells in 24-well plates and transfected with the indicated plasmids, together with AR3-tkk-LUC (a gift from Dr. Paul Rennie), pCMV-3myc-AR, and a cytomegalovirus (CMV)-beta galactosidase (PBL3-beta-gal) construct as a transfection control (a gift from Dr. Shirin Bonni). Luciferase assays were performed as described. Briefly, one day after transfection, cells were washed with phosphate-buffered saline (PBS) and lysed using reporter lysis buffer (Promega). 30 μl of each lysate was transferred into 96-well plates, and luminescence was detected using a Berthold luminometer. Beta-gal staining was used as an internal control.
Chromatin immunoprecipitation (ChIP) and quantitative PCR (qPCR)
The effects of ING3 knockdown on AR recruitment to the FKBP5 ARE were determined by ChIP using 3 × 10
7 C4-2 cells transfected with siCtrl or siING3 for 48 h in media supplemented with 5% CSS, +/– 10 nM MB. Cells were crosslinked using 1% formaldehyde. Cells were lysed in 1 ml ChIP lysis buffer and sonicated for 8 × 12 s. After centrifugation, the supernatants were immunoprecipitated with rabbit anti-AR (N-20, Santa Cruz) or rabbit control IgG overnight at 4 °C and incubated with Protein A Beads (GE Healthcare) for 2 h at 4 °C. Immunoprecipitates were washed with ChIP lysis buffer, with Tris-EDTA (TE) buffer, and then eluted. The IP and input samples were reverse crosslinked using NaCl at 65 °C overnight, and the DNA was isolated. Binding of AR to the Androgen Response Element (ARE) was tested using qPCR. The primer sequences used were FKBP5 ARE6/7: Fwd 5'-CCCCCCTATTTTAATCGGAGTAC-3' and Rev 5'-TTTTGAAGAGCACAGAACACCCT-3', Non-specific Fwd 5'-GGTCAGGTTTTGGTTGAGGA-3' and Rev 5'-CAAGCACAGTGAGGGAGACA-3'. TRIzol and an Omniscript Reverse Transcription kit (Qiagen) were used for isolating total RNA and generating complementary DNA (cDNA). Real-time PCR was performed using Maxima SYBR Green Mastermix (Fermentas) with an Applied Biosystems 7900HT PCR system. The qPCR primer sequences are listed in Additional file
2: Table S1.
Tissue microarray (TMA) study and quantitative analysis
Sections (4-μm thick) were cut from TMA blocks and deparaffinized in xylene, rinsed in ethanol, and rehydrated. Heat-induced epitope retrieval was at 121 °C, pH 6 in Target Retrieval Solution (Dako) for 3 min in a decloaking chamber (Biocare Medical). Slides were stained using a Dako Autostainer. Endogenous peroxidase activity was quenched with peroxidase block (10 min, Dako) followed by a 15-min protein block (Signal Stain, Cell Signaling, Danvers, MA, USA). Slides were washed with Tris-buffered saline with Tween (TBST) and incubated at room temperature for 60 min with Signal Stain protein block containing a 1:1500 dilution of ING3 mouse mAb [
24] and a 1:100 dilution of anti-pan-cytokeratin rabbit polyclonal antibody (Dako). Secondary reagents were incubated at room temperature for 60 min: ready-to-use goat anti-mouse antibody conjugated to a horseradish peroxidase-decorated dextran polymer backbone from the DAKO EnVision + system (Dako) and 1:200 dilution of Alexa-555 conjugated goat anti-rabbit antibody (Invitrogen). Slides were washed with TBST and incubated for 5 min with the Tyramide Signal Amplification (TSA)-Plus Cy5 reagent (Perkin Elmer). After three washes in TBST, slides were mounted with ProLong® Gold anti-fade mounting medium containing DAPI and stored at 4 °C overnight before scanning. For automated image acquisition we used an Aperio Scanscope FL 8/10-bit monochrome TDI line-image capture camera with filters specific for DAPI, Cy3 (Alexa-555) to define the tumor cytosolic compartment based on cytokeratin, and Cy5 for ING3. Images were analyzed using AQUAnalysis® version 2.3.4.1. Scores were based on total percent area positive for ING3.
Immunofluorescence
Cells were grown on coverslips and fixed with 4% paraformaldehyde in PBS and permeabilized using 0.1% Triton X-100 (Millipore) in PBS. Fixed cells were blocked using 5% BSA for 1 h. Ki67 antibody (Dako) or AR antibody (N-20, Santa Cruz) were used at 1:200 in PBS for 2 h, washed with PBS, and incubated with Alexa-488 goat anti-mouse secondary antibody (1:1000 in PBS/5% bovine serum albumin (BSA)) for 1 h. Cells were then mounted on slides and analyzed using an Axiovert 200 microscope.
Cell survival and proliferation assay
LNCaP, PC3, and DU145 cells were transfected with siCtrl or siING3 and 1 × 104 cells were seeded in 24-well plates. The cells were washed twice and stained with 0.1% crystal violet for 15 min at room temperature and washed. Alamar Blue assays were performed to estimate cell proliferation according to the manufacturer's protocol. Cell proliferation was also monitored by seeding cells in 96-well plates in parallel and counting cells at the indicated times using a Celigo Cell Cytometer (Cyntellect).
Anchorage-independent soft agar assay
Agar (0.5%) was prepared in RPMI containing 10% FBS, and 1 ml was poured into each well of 24-well plates to form a bottom layer. 1 × 104 cells were then mixed with RPMI-20% FBS containing 0.3% agarose and poured on top of the bottom layer. Colonies were analyzed using an inverted microscope 10 days after seeding. Colony diameters were measured using ImageJ software, and the colony volumes were calculated (4/3πr3).
Transwell migration assay
Transwell inserts (Corning) were placed in 24-well plates, and 500 μl of RPMI-20% FBS was added to the plate bottoms. We seeded 5 × 104 LNCaP cells on top of the transwell inserts and supplemented with 250 μl of RPMI + CSS or RPMI + 1 nM MB. The inserts were fixed at the indicated time points with 4% paraformaldehyde and methanol and stained with crystal violet. For quantification, six random fields were chosen, and the cells were counted in a single-blinded fashion.
Wound healing assay
C4-2 cells stably infected with shING3 or shCtrl were plated at 80% confluence in 6-well plates. Doxycycline was added to induce the expression of shRNA, and the cells were grown in RPMI supplemented with 10 nM MB for the duration of the experiment. A 200-μl sterile tip was used to wound the monolayers. At the indicated time points, images were taken from the same fields across the course of the experiment. The percentage of healed wound was then calculated using the following formula: 1-(surface area in one field)/(surface area of the same field at day 0).
Statistics
All experiments were done in triplicate. Each patient's tissue samples were punched in duplicate on TMA slides. Graphpad Prism was used for graphs and statistical analyses such as standard error calculations, confidence intervals, Student's t tests and analysis of variance (ANOVA) statistics. SPSS statistics software was used for analyzing the TMA results including Kaplan-Meier and Cox proportional hazard analyses.
Discussion
The androgen receptor (AR) pathway is a major contributor to prostate cancer and, coupled with other oncogenic signaling pathways, plays a key role in the initiation and progression of this disease [
40,
41]. In this study, we have identified ING3 as a novel AR co-activator in PC. Altering ING3 levels in PC cells showed that it positively regulates AR, enhancing effects of androgens on the expression of AR-regulated genes and an ARE-driven reporter. ING3 exerts this effect by promoting AR-TIP60 interaction, thereby increasing the acetylation of AR, its translocation to the nucleus, and activation as a transcription factor. This does not require the ING3 PHD region that interacts specifically with the H3K4Me3 [
19,
20,
42], identifying a novel chromatin-independent role of ING3. Moreover, knockdown of ING3 inhibits PC cell growth, indicating that ING3 plays an oncogenic role in prostate cancer. In contrast to previous studies in other tumor types where ING3 was reported to function as a tumor suppressor, we find that ING3 levels are higher in aggressive PCs, and that a high level of ING3 is a prognostic factor predicting poorer survival in patients with low AR levels. A model for how our data suggest that ING3 functions to activate the AR signaling pathway in PC biology is shown in Fig.
5e.
A carboxyl-terminal deletion mutant of ING3 lacking the PHD (Fig.
2i) interacted with and efficiently activated the AR. This function is likely distinct from epigenetic properties of ING3 since this form of ING3 is incapable of targeting the TIP60 complex to the H3K4Me3 mark. Similar PHD-independent effects for ING family proteins were seen for ING2 during C2C12 myoblast differentiation [
43] and ING4-induced apoptosis in prostate epithelium [
44]. These data indicate two distinct functions for ING3, one in coordinating a cytoplasmic complex to enhance AR acetylation efficiency and another to target the TIP60 complex to chromatin via recognition of H3K4Me3. Altered localization of ING1 in brain cancers [
45] and its shuttling from the cytoplasm to nucleus by interaction with 14-3-3 proteins [
46] are consistent with ING proteins functioning in multiple cell compartments.
Recently, the PHD of ING3 was reported to be essential for DNA damage-induced apoptosis in MCF-7 cells where interaction between the ING3 PHD and the H3K4Me3 mark was reported to be in the submicromolar range [
47]. In contrast to this role, we have identified a chromatin-independent function for both full-length ING3 and a shorter isoform lacking the C-terminal domain. These differential functions of ING3 isoforms are not well defined and require further investigation. Consistent with our observation that ING3 interacts with and targets the TIP60 KAT complex to activate the AR by acetylation, a recent study reported that ING1, a stoichiometric member of the Sin3A KDAC complex [
17] that directs deacetylation activity, functions as an AR co-repressor [
48]. These and other studies, therefore, support the idea that the ING proteins can function to target acetylation and deacetylation activities to the H3K4Me3 mark in chromatin, as well as serve as scaffolding proteins to promote the acetylation or deacetylation of target proteins in the cytoplasm that have major impact on biological processes such as the AR pathway.
AR acetylation is known to be an essential step in AR activation, and increased activity of KATs such as p300 and TIP60 is involved in progression of PC [
8‐
10]. This post-translational modification occurs in the hinge region of AR, leading to nuclear localization signal (NLS) unmasking and nuclear translocation. ING3 promoted TIP60-AR association in the cytoplasm, inducing AR acetylation, nuclear translocation, and activation of target genes, including
FKBP5, an immunophilin that regulates the AR, NF-kB, and the glucocorticoid receptor [
49].
FKBP5, induced by AR via several AREs, modulates the AR pathway through forming a positive feedback loop [
35,
50,
51] as noted in Fig.
5e. While we observed ING3 effects on AR binding to the
FKBP5-ARE, the role of other pathways in regulation of this and other AR-sensitive genes cannot be excluded. The differential effects of ING3 on
FKBP5 as well as other selected androgen-regulated genes in this study underline the complex nature of the regulation of these genes. Indeed, according to the ENCODE data portal, there are several other transcription factors that can occupy the promoters of
PSA,
TMPRSS2, and
FKBP5, the list of which interestingly includes (but is not limited to) other members of the nuclear receptor family. It is therefore likely that the overall effect of ING3 on selected genes can be due to its differential regulation of other transcription factors, independent of its AR-activating function. The interaction of ING3 with the DNA-binding domain of the AR, which is the most conserved domain among nuclear receptors, further suggests other functions beyond the AR; these remain unclear at this point and require additional investigation. This interaction can also be of clinical relevance in prostate cancer, as AR splice variants that lack an intact DNA-binding domain are described as one of the mechanisms promoting CRPC [
52].
When PC cells were grown in an androgen-depleted medium that mimics clinical androgen deprivation therapy (ADT) conditions, ING3 knockdown significantly reduced cell growth. Consistent with this observation, an independent RNA interference-based screen identified ING3 as a positive regulator of proliferation and survival in androgen-deprived VCaP cells [
28]. This is of clinical significance, as resistance to ADT is one of the important challenges in PC management. However, ING3 knockdown also reduced the growth and migration of AR-negative prostate cancer cells, DU145 and PC3, indicating its role in AR-independent pathways. This is likely considering the fact that ING3 and its associated KAT complex are epigenetic regulators with diverse cellular functions [
53]. While we report the novel role of ING3 in AR pathway activation, the effects on global gene regulation and chromatin remodeling, as expected from this class of proteins, should not be overlooked. Indeed, our preliminary observations suggest that ING3 can affect cell cycle pathways through its chromatin binding properties.
In contrast to previous studies in melanoma and hepatocellular carcinoma reporting reduced ING3 levels in aggressive cancers [
54], our analyses of primary prostate tumors showed that high levels of ING3 predict poorer outcome in patients with low AR levels. A similar trend was observed when analyzing recurrence rate using TCGA data stratified based on AR levels (data not shown). This indicates that higher ING3 levels can compensate for low AR levels by activating AR, promoting PC growth. In the context of AR hyperactivation, ING3 may not be required in the process and may primarily function through gene regulation, for example, by reducing apoptosis upon RSK-mediated suppression [
55]. In addition to the effects of androgens and AR antagonists, epithelial-mesenchymal transition (EMT) and invasion of PC cells were recently reported to be dependent on the levels of AR protein, with low levels of AR promoting androgen-induced EMT [
56]. This is in line with our findings that ING3 modulated cell migration and that higher ING3 levels correlated with poorer outcome in the subset of patients having tumors with low levels of AR. We recently found that high levels of ING3 also correlate with poorer survival in ERG-negative PC [
25]. Although the interplay between ERG and AR remains unclear, several studies have suggested that the ERG fusion protein inhibits AR expression and activity at several loci [
57], supporting the idea that ING3 can potentiate the activity of the AR pathway, particularly when AR inhibitory factors such as ERG are absent. Together, these data identify ING3 as a proto-oncogene in PC by regulating the AR pathway through acetylation, and identify it as a novel prognostic biomarker for primary prostate cancer.
Acknowledgements
We thank Donna Boland of the University of Calgary Hybridoma Facility for antibodies and Dr. Emeka Enwere and Michelle Dean for TMA-AQUA expertise. We thank the live cell imaging facility at the Snyder Institute for Chronic Diseases and Dr. Pina Colarusso and Rima-Marie Wazen for their assistance. We thank Dr. Tak Fung for statistical assistance and Dr. Craig Robson for AR deletion constructs. The C4-2 cell line was a kind gift from Dr. Martin Gleave, the pCMV-3myc-AR plasmid was a gift from Dr. Marja Nevalainen, pCIN4-FLAG-HA-TIP60 was a gift from Dr. Wei Gu, pCDNA3-ARK630R and ARK632/33R were gifts from Dr. Richard Pestell, AR3-tkk-LUC was a gift from Dr. Paul Rennie, and the PBL3-beta-gal construct was a gift from Dr. Shirin Bonni.