ING3 promotes prostate cancer growth by activating the androgen receptor

Background The androgen receptor (AR) is a major driver of prostate cancer, and increased AR levels and co-activators of the receptor promote the development of prostate cancer. INhibitor of Growth (ING) proteins target lysine acetyltransferase or lysine deacetylase complexes to the histone H3K4Me3 mark of active transcription, to affect chromatin structure and gene expression. ING3 is a stoichiometric member of the TIP60 lysine acetyltransferase complex implicated in prostate cancer development. Methods Biopsies of 265 patients with prostate cancer were stained for ING3, pan-cytokeratin, and DNA. LNCaP and C4-2 androgen-responsive cells were used for in vitro assays including immunoprecipitation, western blotting, Luciferase reporter assay and quantitative polymerase chain reaction. Cell viability and migration assays were performed in prostate cancer cell lines using scrambled siRNA or siRNA targeting ING3. Results We find that ING3 levels and AR activity positively correlate in prostate cancer. ING3 potentiates androgen effects, increasing expression of androgen-regulated genes and androgen response element-driven reporters to promote growth and anchorage-independent growth. Conversely, ING3 knockdown inhibits prostate cancer cell growth and invasion. ING3 activates the AR by serving as a scaffold to increase interaction between TIP60 and the AR in the cytoplasm, enhancing receptor acetylation and translocation to the nucleus. Activation is independent of ING3's ability to target the TIP60 complex to H3K4Me3, identifying a previously unknown chromatin-independent cytoplasmic activity for ING3. In agreement with in vitro observations, analysis of The Cancer Genome Atlas (TCGA) data (n = 498) and a prostate cancer tissue microarray (n = 256) show that ING3 levels are higher in aggressive prostate cancers, with high levels of ING3 predicting shorter patient survival in a low AR subgroup. Including ING3 levels with currently used indicators such as the Gleason score provides more accurate prognosis in primary prostate cancer. Conclusions In contrast to the majority of previous reports suggesting tumor suppressive functions in other cancers, our observations identify a clear oncogenic role for ING3, which acts as a co-activator of AR in prostate cancer. Data from TCGA and our previous and current tissue microarrays suggest that ING3 levels correlate with AR levels and that in patients with low levels of the receptor, ING3 level could serve as a useful prognostic biomarker

The p53 Tumor Suppressor Is Stabilized by Inhibitor of Growth 1 (ING1) by Blocking Polyubiquitination

The INhibitor of Growth tumor suppressors (ING1-ING5) affect aging, apoptosis, DNA repair and tumorigenesis. Plant homeodomains (PHD) of ING proteins bind histones in a methylation-sensitive manner to regulate chromatin structure. ING1 and ING2 contain a polybasic region (PBR) adjacent to their PHDs that binds stress-inducible phosphatidylinositol monophosphate (PtIn-MP) signaling lipids to activate these INGs. ING1 induces apoptosis independently of p53 but other studies suggest proapoptotic interdependence of ING1 and p53 leaving their functional relationship unclear. Here we identify a novel ubiquitin-binding domain (UBD) that overlaps with the PBR of ING1 and shows similarity to previously described UBDs involved in DNA damage responses. The ING1 UBD binds ubiquitin with high affinity (Kd∼100 nM) and ubiquitin competes with PtIn-MPs for ING1 binding. ING1 expression stabilized wild-type, but not mutant p53 in an MDM2-independent manner and knockdown of endogenous ING1 depressed p53 levels in a transcription-independent manner. ING1 stabilized unmodified and six multimonoubiquitinated forms of wild-type p53 that were also seen upon DNA damage, but not p53 mutants lacking the six known sites of ubiquitination. We also find that ING1 physically interacts with herpesvirus-associated ubiquitin-specific protease (HAUSP), a p53 and MDM2 deubiquitinase (DUB), and knockdown of HAUSP blocks the ability of ING1 to stabilize p53. These data link lipid stress signaling to ubiquitin-mediated proteasomal degradation through the PBR/UBD of ING1 and further indicate that ING1 stabilizes p53 by inhibiting polyubiquitination of multimonoubiquitinated forms via interaction with and colocalization of the HAUSP-deubiquitinase with p53

Involvement of caspase 1 and its activator Ipaf upstream of mitochondrial events in apoptosis

PTP-S2/TC45 is a nuclear protein tyrosine phosphatase that activates p<SUP>53</SUP> and induces caspase 1-dependent apoptosis. We analyzed the role of ICE protease-activating factor (Ipaf), an activator of caspase 1 in p<SUP>53</SUP>-dependent apoptosis. We also determined the sequence of events that lead to apoptosis upon caspase 1 activation by Ipaf. PTP-S2 expression induced Ipaf mRNA in MCF-7 cells which was dependent on p<SUP>53</SUP>. PTP-S2-induced apoptosis was inhibited by a dominant-negative mutant of Ipaf and also by an Ipaf-directed short-hairpin RNA. Doxorubicin-induced apoptosis was potentiated by the expression of caspase 1 (but not by a catalytic mutant of caspase 1) and required endogenous Ipaf. Doxorubicin treatment of MCF-7 cells resulted in activation of exogenous caspase 1, which was partly dependent on endogenous Ipaf. An activated form of Ipaf induced caspase 1-dependent apoptosis that was inhibited by Bcl2 and also by a dominant inhibitor of caspase 9 (caspase 9s). Caspase 1-dependent apoptosis induced by doxorubicin was also inhibited by Bcl2 and caspase 9s, but caspase 1 activation by activated Ipaf was not inhibited by Bcl2. Mitochondrial membrane permeabilization was induced by caspase 1 and activated Ipaf, which was inhibited by Bcl2, but not by caspase 9s. Expression of caspase 1 with activated Ipaf resulted in the activation of Bax at mitochondria. Our results suggest that Ipaf is involved in PTP-S2-induced apoptosis and that caspase 1, when activated by Ipaf, causes release of mitochondrial proteins (cytochrome c and Omi) through Bax activation, thereby functioning as an initiator caspase

The ING1a Tumor Suppressor Regulates Endocytosis to Induce Cellular Senescence Via the Rb-E2F Pathway

<div><p>The INhibitor of Growth (ING) proteins act as type II tumor suppressors and epigenetic regulators, being stoichiometric members of histone acetyltransferase and histone deacetylase complexes. Expression of the alternatively spliced ING1a tumor suppressor increases >10-fold during replicative senescence. ING1a overexpression inhibits growth; induces a large flattened cell morphology and the expression of senescence-associated β-galactosidase; increases Rb, p16, and cyclin D1 levels; and results in the accumulation of senescence-associated heterochromatic foci. Here we identify ING1a-regulated genes and find that ING1a induces the expression of a disproportionate number of genes whose products encode proteins involved in endocytosis. Intersectin 2 (ITSN2) is most affected by ING1a, being rapidly induced >25-fold. Overexpression of ITSN2 independently induces expression of the p16 and p57^KIP2 cyclin-dependent kinase inhibitors, which act to block Rb inactivation, acting as downstream effectors of ING1a. ITSN2 is also induced in normally senescing cells, consistent with elevated levels of ING1a inducing ITSN2 as part of a normal senescence program. Inhibition of endocytosis or altering the stoichiometry of endosome components such as Rab family members similarly induces senescence. Knockdown of ITSN2 also blocks the ability of ING1a to induce a senescent phenotype, confirming that ITSN2 is a major transducer of ING1a-induced senescence signaling. These data identify a pathway by which ING1a induces senescence and indicate that altered endocytosis activates the Rb pathway, subsequently effecting a senescent phenotype.</p> </div

Regulation of endocytosis by ING1a.

<p>(A) Cells infected with adenovirus-GFP or adenovirus-GFP-ING1a (with GFP and ING1a expressed under separate promoters) were serum-deprived for 12 h, stimulated with 100 ng/ml EGF, and fixed 15 or 180 min later. Indirect immunofluorescence for EGFR showed reduced amounts of internalized EGFR in cells expressing ING1a at early time points but persistence of the EGFR at later times. All cells were treated with 10 µg/ml of cycloheximide to inhibit de novo protein synthesis. We took 0- and 15-min images using 63× objectives, while 180 min were imaged at 40× magnification. (B) Biotin internalization assay. Ad-GFP-ING1a and Ad-GFP-expressing cells were serum-starved and EGF-stimulated for the indicated times. Total cell surface proteins were biotinylated, and EGFR was immunoprecipiatated. Biotinylated cell surface EGFR at the indicated time points was detected using streptavidin-HRP. The graph shows the rate of EGFR internalization in GFP- and ING1a-expressing cells as estimated by scanning densitometry. (C) A431 cells infected with adenovirus-GFP/ING1a were serum-starved and checked for tyrosine phosphorylation of the immunoprecipiated EGFR upon EGF stimulation for the indicated time-points. (D) Cells left untreated or expressing GFP or GFP plus ING1a for 48 h were serum-starved overnight (12 h) and stimulated with 100 ng/ml EGF for the indicated times. 100 µg/ml of cycloheximide was used to inhibit protein synthesis. Cells were lysed and levels of EGFR were analyzed by western blotting. Actin was used as a loading control. (E) Wild-type or ING1 knockout MEFs were serum-starved overnight, treated with 10 µg/ml of cycloheximide for 20 min, stimulated with 100 ng/ml EGF and were harvested at the indicated times. Proteins were resolved by SDS-PAGE and blotted with α-EGFR antibody and α-actin as loading control. (F) mRNA levels of Ese2, the ITSN2 mouse homologue in MEF WT and ING^−/− cells. RNA was isolated from these cells, and qRT-PCR was performed on three independent replicates. The gene expression levels were normalized to GAPDH (<i>p</i><0.05).</p

Gene expression in response to ING1a.

<p>(A) Functional categorization of 242 genes reproducibly up-regulated in response to ING1a overexpression. Thirty-five percent of all up-regulated genes and 40% of those induced >2.5-fold had functions in endocytosis, trafficking, and associated signaling pathways. (B) Quantitative PCR (qPCR) validation of mRNA levels of a representative set of genes up- and down-regulated in ING1a overexpressing cells. Bars represent standard deviations of three independent trials (<i>p</i><0.05).</p

ITSN2 knockdown ameliorates ING1a-induced senescence phenotypes.

<p>(A) Biotin internalization assay in A431 cells expressing Ad-ING1a+Control siRNA or ITSN2 siRNA. Cells were serum-starved overnight and stimulated with EGF for the indicated times. Biotinylated cell surface EGFR was quantified using scanning densitometry. Results of three trials are shown in the graph. (B) A β-gal assay was carried out in cells expressing GFP and ING1a after transfection with control or ITSN2 siRNA. (C) Hs68 cells expressing Ad-GFP, Ad-ING1a, or Ad-ING1a together with control siRNA or ITSN2 siRNA were analysed for proliferation using a BrdU incorporation assay. The percentage of cells that incorporated BrdU is presented in the histogram (<i>p</i><0.05). (D) Low passage (young) Hs68 cells were transfected with control siRNA (50 nM) or ITSN2 smartpool siRNA (50 nM). Twenty-four hours later cells were infected with Ad-GFP or Ad-ING1a-expressing viruses, and 48 h later the levels of ITSN2, p16, RB, and HSP70 mRNA were measured by quantitative real-time PCR using gene-specific primers. The mRNA levels were normalized to β-actin levels. (E) Senescent Hs68 cells (MPD80) were transfected with control siRNA or ITSN2 siRNA and checked for the relative levels of the indicated mRNAs using quantitative real-time PCR. The values were normalized to actin. (F) Relative mRNA levels of representative E2F target genes in ING1a-expressing cells transfected with control siRNA or ITSN2 siRNA. The values are plotted after normalizing to actin (<i>p</i><0.06).</p

ITSN2 expression precedes the appearance of senescence markers.

<p>(A) RNA from uninfected, GFP, or GFP-ING1a-expressing Hs68 cells was isolated at 12, 24, 36, and 48 h post-virus infection. Levels of ING1a mRNA were checked to confirm that ING1a was overexpressed at each of the indicated time points (top most panel). Induction of ITSN2, EPS15, p16, and Rb were checked at these time points by qRT-PCR using gene-specific primers. Arrows indicate the time points at which the induction of expression of each of these genes were noted. (B) Binding of ING1a to the ITSN2 promoter was tested by chromatin immunoprecipitation assay. Binding was not seen in the control GFP-expressing cells. The lower panel is the schematic representation of the location of the primer sequences in the upstream region of ITSN2 that were used in this study. Mouse IgG was used as a nonspecific antibody control. PCNA and Cyclin A promoter regions were used as positive and negative controls for the ChIP assay, respectively. (C) ITSN2 overexpression for 48 h in young fibroblasts resulted in formation of senescent-associated heterochromatin foci, containing the heterochromatin protein 1 (HP1γ), similar to those seen in response to ING1a. Cells also showed significant SA-beta gal staining in response to ING1a and ITSN2.</p

Gene expression levels and kinetics of endocytosis in senescing cells.

<p>(A) mRNA levels of ITSN2, EPS15, HSP70, and JAK2 were compared in young and old fibroblasts. Levels were normalized to GAPDH. Levels of p16 and ING1a mRNA serve as controls to confirm the senescent state of cells. (B) EGFR degradation was examined in low passage and senescing cells. The samples were prepared as described previously, and EGFR amounts were estimated by western blotting. EGFR persisted in senescent compared to young cells, and ING1a was expressed at much higher levels in senescent cells as previously reported <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001502#pbio.1001502-Soliman1" target="_blank">[23]</a>. (C) mRNA levels of ITSN2 and other endocytic genes in cells knocked down for ING1a using siRNA (<i>p</i><0.05). (D) ING1a and ITSN2 levels in other forms of stress induced premature senescence (SIPS) using doxorubicin and t-butyl hydroperoxide. P16 and p21 are cyclin-dependent kinase inhibitors that serve as senescence markers, and HSP70 as a stress marker. The top panels show SA-β-gal staining as a marker for senescence induction in response to the treatments noted.</p