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

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

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

ING1a delays growth factor signaling, delays Rb phosphorylation, and inhibits E2Factivation.

<p>(A) Low passage (young) Hs68 fibroblasts (MPD 25) infected with Ad-GFP or Ad-ING1a for 24 h were serum-starved and stimulated with 100 ng/ml of EGF for the indicated time points. Western blots of p-Src, p-Erk, p-Akt, p-38MAPK, and p-Rb were performed to check their activation status as estimated by their phosphorylation. The same lysates were also probed with antibodies for total Src, Akt, p38MAPK, and Rb. Actin was used as the internal loading control. (B) Hs68 cells synchronized by serum starvation for 16 h were released in the presence of Ad-GFP or Ad-ING1a and checked for the phosphorylation status of Rb at indicated time points. (C) Hs68 cells synchronized by serum starvation for 24 h were released in complete medium in the presence of Ad-GFP or Ad-ING1a. Twenty-four hours later, cells were harvested and lysates were immunoprecipiated using anti-Rb antibody. Immunoprecipitates were electrophoresed and blotted with the indicated antibodies. Levels of E2F, Rb, actin, and ING1a were checked in whole cell lysates. (D) Relative mRNA levels of E2F target genes in cells expressing Ad-GFP or Ad-ING1a. The values were normalized to actin (<i>p</i><0.05). (E) Total RNA from cells transfected with ITSN2 was isolated and the expression of p16, p57^KIP2, and Rb were examined. All values were normalized to levels seen in cells transfected with the same amount of control GFP plasmid, which was also used to monitor and control for transfection efficiency.</p

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

Genes (A) up-regulated or (B) down-regulated at least 2.5-fold in response to ING1a overexpression in primary Hs68 fibroblasts.

<p>Cells expressing GFP were used as a negative control. Seven of the 17 most highly induced genes (>40%) highlighted by asterisks have characterized functions in endocytosis, signaling, and trafficking.</p

Model for ING1a-induced senescence via ITSN2 and Rb.

<p>Increased levels of ING1a, produced by alternative splicing in response to short telomeres or oxidative stress, bind and activate the promoter of ITSN2, increasing its levels. This leads to inhibition of endocytosis and a loss of signal transduction via clathrin- and caveolae-mediated endocytosis. This increases the levels of p16 and of p57^KIP2, which inhibit Rb phosphorylation, maintaining it in its hypophosphorylated, growth-inhibitory state. This and the ITSN2-independent induction of Rb by ING1a result in the accumulation of high levels of active Rb that inhibits E2F, blocking the expression of most E2F target genes. Inability to induce transcription of growth-promoting genes inhibits cell cycle progression and, combined with Rb-induced accumulation of SAHF, results in senescence. We propose that this mechanism also acts during normal replicative senescence, since the levels of both ING1a and intersectin 2 are dramatically increased in high passage senescing cells. Dashed arrows represent the ING1a-mediated senescence pathway identified in this study.</p

Additional file 1: Figure S1. of Stromal ING1 expression induces a secretory phenotype and correlates with breast cancer patient survival

Quantitation of 3-D culture colony morphological changes. Colony images from 3-D cultures of HMF3s cells expressing GFP + MCF7 cells and HMF3s cells expressing ING1a + MCF7 cells were visually scored for their levels of disorganization and aggressiveness as estimated by divergence from uniformity (** p < 0.001). (DOC 58 kb