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Replicative capacity of normal human cells decreases as telomeric sequence is lost at each division. It is believed that when a subset of chromosomes reach a critically short length, an ATM-initiated and p53-mediated transcriptional response inhibits cell growth, promoting cell senescence. In addition to loss of telomeric sequence, senescence can be induced by other stresses including ionizing radiation, oxidative damage, chemical crosslinkers like the chemotherapeutic agent cisplatin, as well as overactivation of oncogenes and tumor suppressors. Our group found that the expression of an isoform of the INhibitor of Growth 1 gene called ING1a increases approximately 10-fold as fibroblasts approach senescence and that forced expression rapidly induces a senescent phenotype in primary diploid fibroblasts, epithelial and endothelial cells that resembles replicative senescence by most physical and biochemical measures. ING1a induces these changes through strongly inhibiting endocytosis to block mitogen signaling by inducing the expression of intersectin 2, a key scaffolding protein of the endosomal pathway. This, in turn increases the expression of Rb and of p57Kip2 and p16INK4a that serve to maintain Rb is an active, growth inhibitory state. The ING1a model is currently being used to better understand the mechanism(s) responsible for activating Rb to enforce the senescent state.

Simple Summary Gene regulation is the major mechanism that allows us to develop from a single cell to a fully formed adult body containing numerous organs and tissues, >200 cell types and a total of about 50 trillion cells. One of the ways our cells turn genes “on and off” is through the addition or removal of acetyl moieties (CH3CO) to proteins called histones. Our DNA is stabilized and compacted by being wrapped around groups of 8 histones to form nucleosomes. The addition of acetyl groups to histones loosens that wrapping, allowing the DNA to be more accessible for transcription, “turning on” genes of interest. This process is coordinated by enzymes called histone acetyltransferases (HATs, also called lysine acetyltransferases) and histone deacetylases (HDACs or lysine deacetylases). The precise coordination of these enzyme activities is essential to allow our stem cell populations to replenish themselves or differentiate along different pathways. Many of these enzymes have been described as being key regulators for hematopoietic, brain, and mesenchymal stem cells. This review describes how HATs and HDACs regulate stem cell processes and what is currently known regarding the roles of acetylation of histones in stem cell biology. Abstract Acetylation of histones is a key epigenetic modification involved in transcriptional regulation. The addition of acetyl groups to histone tails generally reduces histone-DNA interactions in the nucleosome leading to increased accessibility for transcription factors and core transcriptional machinery to bind their target sequences. There are approximately 30 histone acetyltransferases and their corresponding complexes, each of which affect the expression of a subset of genes. Because cell identity is determined by gene expression profile, it is unsurprising that the HATs responsible for inducing expression of these genes play a crucial role in determining cell fate. Here, we explore the role of HATs in the maintenance and differentiation of various stem cell types. Several HAT complexes have been characterized to play an important role in activating genes that allow stem cells to self-renew. Knockdown or loss of their activity leads to reduced expression and or differentiation while particular HATs drive differentiation towards specific cell fates. In this study we review functions of the HAT complexes active in pluripotent stem cells, hematopoietic stem cells, muscle satellite cells, mesenchymal stem cells, neural stem cells, and cancer stem cells.

The human genome's spectrum of gene expression patterns is vast and, while constantly adapting to surrounding cues, versatile and variable. It generates hundreds of different cell types forming multiple tissues and organ systems [1], amongst which well over a hundred cancer types have been identified to date [1]. While originating from the same ancestor, thereby harboring nearly identical genetic information, every eukaryotic cell type presents with its own distinguishable biological behavior and phenotype, underscoring the degree of cell plasticity inherent during development. Instead of being established by DNA sequence alterations, the rich diversity of cell identities is now thought to be defined and largely driven by epigenetic variation—a regulatory engine fueled by the homeostatic balance of chromatin regulators and designed to maintain physiological organismal homeostasis to ensure functional gene expression in both stressful and quiescent situations. Disruption of chromatin homeostasis results in it becoming either aberrantly restrictive or aberrantly permissive, thereby driving tumor initiation in pre-malignant cells and/or tumor evolution and adaptation in malignant cells. Hence, epigenetic instability may represent the 11th hallmark of cancer—both as a cause and consequence of, or even without concomitant genomic instability.

Telomeres are protective structures, composed of nucleic acids and a complex protein mixture, located at the end of the chromosomes. They play an important role in preventing genomic instability and ensuring cell health. Defects in telomere integrity result in cell dysfunction and the development of diseases, including neurodegenerative disorders, cancer and premature aging syndromes, among others. Loss of telomere integrity during normal cell aging also initiates DNA damage signals that culminate in the senescence phenotype. Fluorescence microscopy has allowed researchers to study the dynamics, shape, localization, and co-distribution of telomeres with proteins of interest. The microscopy tools to investigate these structures have evolved, making it possible to understand in greater detail the molecular mechanisms affecting telomeres that contribute to cell aging and the development of age-related diseases. Using human fibroblasts as an example, we will highlight several characteristics of telomeres that can be investigated using three different microscopy systems, including wide-field microscopy, and the two super-resolution techniques called 3D Structured Illumination Microscopy (3D-SIM) and direct Stochastic Optical Reconstruction Microscopy (dSTORM). In this review, we will also discuss their limitations and highlight their importance in answering telomere-related scientific questions.

Stem cell-like brain tumor initiating cells (BTICs) cause recurrence of glioblastomas, with BTIC ‘stemness’ affected by epigenetic mechanisms. The ING family of epigenetic regulators (ING1-5) function by targeting histone acetyltransferase (HAT) or histone deacetylase complexes to the H3K4me3 mark to alter histone acetylation and subsequently, gene expression. Here we find that ectopic expression of ING5, the targeting subunit of HBO1, MOZ and MORF HAT complexes increases expression of the Oct4, Olig2 and Nestin stem cell markers, promotes self-renewal, prevents lineage differentiation and increases stem cell pools in BTIC populations. This activity requires the plant homeodomain region of ING5 that interacts specifically with the H3K4me3 mark. ING5 also enhances PI3K/AKT and MEK/ERK activity to sustain self-renewal of BTICs over serial passage of stem cell-like spheres. ING5 exerts these effects by activating transcription of calcium channel and follicle stimulating hormone pathway genes. In silico analyses of The Cancer Genome Atlas data suggest that ING5 is a positive regulator of BTIC stemness, whose expression negatively correlates with patient prognosis, especially in the Proneural and Classical subtypes, and in tumors with low SOX2 expression. These data suggest that altering histone acetylation status and signaling pathways induced by ING5 may provide useful clinical strategies to target tumor resistance and recurrence in glioblastoma.

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. Electronic supplementary material The online version of this article (doi:10.1186/s12916-017-0854-0) contains supplementary material, which is available to authorized users.

Purpose: The current study was designed to investigate the combined influence of ataxia telangiectasia mutated (ATM) and Ki67 on clinical outcome in early stage hormone receptor positive breast cancer (ES-HPBC), particularly in patients with smaller tumors (< 4 cm) and fewer than four positive lymph nodes. Methods: Formalin-fixed paraffin-embedded specimens of resected primary breast tumors from 532 patients diagnosed with early stage breast cancer were used to construct a tissue microarray. Samples from 297 patients were suitable for final statistical analysis. We detected ATM and Ki67 proteins using immunohistochemistry and quantified their expression with digital image analysis. Data on expression levels were subsequently correlated with clinical outcome. Results: Remarkably, ATM expression was useful to stratify the low Ki67 group into subgroups with better or poorer prognosis. Specifically, in the low Ki67 subgroup defined as having smaller tumors and no positive nodes, patients with high ATM expression showed better outcome than those with low ATM, with estimated survival rates of 96% and 89% respectively at 15 years follow up (p = 0.04). Similarly, low-Ki67 patients with smaller tumors, 1-3 positive nodes and high ATM also had significantly better outcomes than their low ATM counterparts, with estimated survival rates of 88% and 46% respectively (p=0.03) at 15 years follow up. Multivariable analysis indicated that the combination of high ATM and low Ki67 is prognostic of improved survival, independent of tumor size, grade, and lymph node status (p = 0.02). Low Ki67/high ATM expression independently predicts favorable disease survival in a multivariate model in low Ki67 subgroup of ES-HPBC (Stage I-III)Variablesp valueHR (95%CI)Low Ki67/high ATM vs Low Ki67/low ATM0.020.36 (0.15-0.88)ize (T1/2 vs T3/4)0.010.16 (0.04-0.69)LN status (- vs +)0.140.49 (0.20-1.25)LVI (_- vs +)0.160.51 (0.20-1.31)Grade (1/2 vs 3)65)0.010.26 (0.10-0.74) Conclusions: These data suggest that the prognostic value of Ki67 can be improved by analyzing ATM expression in ES-HPBC. Citation Format: Feng X, Li H, Kornarga E, Dean M, Lees-Miller S, Riabowol K, Magliocco A, Morris D, Watson P, Enwere E, Bebb G, Paterson A. Refining the prognostic value of Ki67 biomarker by ataxia telangiectasia mutated (ATM) status in a retrospective study of early stage hormone receptor positive breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P2-05-33.

Telomere length and dynamics are central to understanding cell aging, genomic instability and cancer. Currently, there are limited guidelines for analyzing telomeric features in 3D using different cellular models. Image processing for telomere analysis is of increasing interest in many fields, however a lack of standardization can make comparisons and reproducibility an issue. Here we provide a user's guide for quantitative immunofluorescence microscopy of telomeres in interphase cells that covers image acquisition, processing and analysis. Strategies for determining telomere size and number are identified using normal human diploid Hs68 fibroblasts. We demonstrate how to accurately determine telomere number, length, volume, and degree of clustering using quantitative immunofluorescence. Using this workflow, we make the unexpected observation that hTERT-immortalized Hs68 cells with longer telomeres have fewer resolvable telomeres in interphase. Rigorous quantification indicates that this is due to telomeric clustering, leading to systematic underestimation of telomere number and overestimation of telomere size., Nancy Adam et al present a pipeline to analyze interphase telomeres in 3D using wide-field microscopy and image analysis. Illustrating the utility of the workflow, they detect differences in telomere length and clustering in Hs68 fibroblasts depending on whether or not cells are immortalized through hTERT expression.

The ING3 candidate tumour suppressor belongs to a family of histone modifying proteins involved in regulating cell proliferation, senescence, apoptosis, chromatin remodeling, and DNA repair. It is a stoichiometric member of the minimal NuA4 histone acetyl transferase (HAT) complex consisting of EAF6, EPC1, ING3, and TIP60. This complex is responsible for the transcription of an essential cascade of genes involved in embryonic development and in tumour suppression. ING3 has been linked to head and neck and hepatocellular cancers, although its status as a tumour suppressor has not been well established. Recent studies suggest a pro-metastasis role in prostate cancer progression. Here, we describe a transgenic mouse strain with insertional mutation of an UbC-mCherry expression cassette into the endogenous Ing3 locus, resulting in the disruption of ING3 protein expression. Homozygous mutants are embryonically lethal, display growth retardation, and severe developmental disorders. At embryonic day (E) 10.5, the last time point viable homozygous embryos were found, they were approximately half the size of heterozygous mice that develop normally. µCT analysis revealed a developmental defect in neural tube closure, resulting in the failure of formation of closed primary brain vesicles in homozygous mid-gestation embryos. This is consistent with high ING3 expression levels in the embryonic brains of heterozygous and wild type mice and its lack in homozygous mutant embryos that show a lack of ectodermal differentiation. Our data provide direct evidence that ING3 is an essential factor for normal embryonic development and that it plays a fundamental role in prenatal brain formation.

ING2 (Inhibitor of Growth 2) is a tumor-suppressor gene involved in chromatin acetylation whose loss has been observed in nearly 60% of NSCLC. The loss of ING2 expression has been associated with the progression of tumor cells from G1 to S phase, replication stress and genomic instability. The intra-S and the G2-M phase checkpoints are mainly regulated by the cell cycle tyrosine kinase WEE1. Therefore, we hypothesized that the loss of ING2 expression could sensitize NSCLC to the WEE1 inhibitor MK-1175, according to a synthetic lethality model. In silico analysis of the cancer gene dependency map (shRNAi Achilles library v.2016) was used to explore gene interactions with ING2. Antiproliferative effects of drugs were tested in vitro with AlamarBlue and MTT assays in a panel of NSCLC cells lines with varied mutational status. Multiple functional approaches were taken to decrease ING2 expression in RAS-mutated NSCLC cell lines (si-RNA, LNA-gapmer, and CrispR-Cas9). Pharmacologic inhibition of ING2 epigenetic functions was achieved using the HDAC inhibitor SAHA. Cell cycle and apoptosis analysis were performed using flow cytometry according to a BrdU-Pi and Annexin V-PI protocols, respectively. Compusyn software was used to model MK-1775 and SAHA drug interaction. NSCLC cell lines with mutant RAS that expressed high levels of ING2 protein were more resistant to the antiproliferative effect of MK-1775 than similar lines with low levels of ING2. Colony formation assays were consistent with these results. Multiple functional assays reproducibly showed that downregulation of ING2 expression increased MK-1775 sensitivity in vitro. Similar results were obtained with pharmacologic disruption of ING2 from the HDAC-SIN3A complex using SAHA, where disruption conferred a higher antiproliferative effect to MK-1775. Interestingly, combination treatment induced cell cycle progression and synergic cell death through apoptosis in RAS-mutated cell lines nondeficient for ING2. Since the synergistic effect of SAHA and WEE1 inhibitor was shown to be dependent on CHK1 activity in a previous report, we checked whether ING2 and CHK1 expression could be linked. Using public database and functional assays, we indeed find that ING2 downregulation represses CHK1 transcription through a mechanism that remains to be elucidated. Collectively, these findings demonstrate that inhibition of WEE1 by MK-1775 could represent a therapeutic approach to more effectively target ING2-deficient RAS-mutated NSCLC. Moreover, HDAC inhibitors targeting ING2 epigenetic functions synergize with MK-1775, possibly through regulation of CHK1 transcription. Consequently, this combination therapy could also be a promising strategy in ING2-proficient RAS-mutated NSCLC. Citation Format: Charles Ricordel, Subash Thalappilly, Jérome Archambeau, Angela Chan, Nancy Nixon, Benoît Desrues, Gwyn Bebb, Karl Riabowol, Rémy Pedeux. ING2 loss sensitizes KRAS-mutated NSCLC to WEE1 inhibition through regulation of CHK1 expression [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr B06.

The TP53-MDM2-AR-AKT signalling network plays a critical role in the development and progression of prostate cancer. However, the molecular mechanisms regulating this signalling network are not completely defined. By conducting transcriptome analysis, denaturing immunoprecipitations and immunopathology, we demonstrate that the TP53-MDM2-AR-AKT cross-talk is regulated by the deubiquitinating enzyme USP12 in prostate cancer. Our findings explain why USP12 is one of the 12 most commonly overexpressed cancer-associated genes located near an amplified super-enhancer. We find that USP12 deubiquitinates MDM2 and AR, which in turn controls the levels of the TP53 tumour suppressor and AR oncogene in prostate cancer. Consequently, USP12 levels are predictive not only of cancer development but also of patient’s therapy resistance, relapse and survival. Therefore, our findings suggest that USP12 could serve as a promising therapeutic target in currently incurable castrate-resistant prostate cancer.

To assess oncogenic potential, classical transformation assays are based on cell line models. However, cell line based models do not reflect the complexity of human tissues. We thus developed an inducible expression system for gene expression in ex vivo human tissues, which maintain native tissue architecture, such as epithelia and stroma. To validate the system, we transduced and expressed known tumor suppressors (p53, p33ING1b), oncoproteins (RasV12, p47ING3), or controls (empty vector, YFP) in ex vivo prostate tissues, then assessed proliferation by immunohistochemistry of markers (H3S10phos). Herein, we describe how to generate lentiviral vectors and particules, successfully transduce human prostate tissues, induce exogenous gene expression, and assess cellular proliferation.

Cell senescence contributes to organismal aging and is induced by telomere erosion and an ensuing DNA damage signal as cells reach the end of their replicative lifespan in vitro or in vivo. Stresses induced by oncogene or tumor suppressor hyperactivation, oxidative stress, ionizing radiation and other DNA damaging agents result in forms of stress induced premature senescence (SIPS) that show similarities to replicative senescence. Since replicative senescence and SIPS occur over many days and many population doublings of the mass cultures of primary cells used to study senescence, the sequence of events that occur downstream of senescence signaling can be challenging to define. Here we compare a new model of ING1a-induced senescence with several other forms of senescence. The ING1a epigenetic regulator synchronously induces senescence in mass cultures several-fold faster than all other agents, taking 24 and 36 hours to activate the Rb/ p16INK4a, but not the p53 tumor suppressor axis to efficiently induce senescence. ING1a induces expression of intersectin 2, a scaffold protein necessary for endocytosis, altering the stoichiometry of endocytosis proteins, subsequently blocking growth factor uptake leading to activation of Rb signaling to block cell growth. ING1a acts as a novel link in the activation of the Rb pathway that can impose senescence in the absence of activating p53-mediated DNA damage signaling, and should prove useful in defining the molecular events contributing to Rb-induced senescence.

Members of the INhibitor of Growth (ING) family of proteins act as readers of the epigenetic code through specific recognition of the trimethylated form of lysine 4 of histone H3 (H3K4Me3) by their plant homeodomains. The founding member of the family, ING1, was initially identified as a tumor suppressor with altered regulation in a variety of cancer types. While alterations in ING1 and ING4 levels have been reported in a variety of cancer types, little is known regarding ING3 protein levels in normal or transformed cells due to a lack of reliable immunological tools. In this study we present the characterization of a new monoclonal antibody we have developed against ING3 that specifically recognizes human and mouse ING3. The antibody works in western blots, immunofluorescence, immunoprecipitation and immunohistochemistry. Using this antibody we show that ING3 is most highly expressed in small intestine, bone marrow and epidermis, tissues in which cells undergo rapid proliferation and renewal. Consistent with this observation, we show that ING3 is expressed at significantly higher levels in proliferating versus quiescent epithelial cells. These data suggest that ING3 levels may serve as a surrogate for growth rate, and suggest possible roles for ING3 in growth and self renewal and related diseases such as cancer.

Background: The ING family of type II tumour suppressors serve as both epigenetic ‘readers' and target histone acetyl transferase (HAT) and histone deacetylase (HDAC) ‘writers' of the epigenetic histone code. The ING1 protein has also been implicated in regulating microRNA (miRNA) levels. In this study, we identify a link between ING1b and the miRNA epigenetic network. Methods: Primary fibroblasts infected with adenoviruses expressing GFP control or GFP plus ING1b were examined for alterations in miRNA profiles using a miRNA PCR array. Additional experiments confirmed specificity and consequences of altered miRNA expression. Results: MicroRNAs miR-203, miR-375, miR-449b and miR-200c were increased by ING1b overexpression. Ectopic expression of miR-203 inhibited U2OS and MDA-MB-231 cancer cell growth, and induced G1 cell cycle arrest in U2OS cells as estimated by flow cytometry. Transfection with miR-203 inhibitor reversed the proliferation inhibition induced by ING1b in U2OS cells. CHIP assays showed that ING1b bound to the promoter of miR-203. Western blot analyses showed that CDK6, c-Abl and Src were downregulated by the transfection of miR-203. Conclusion: These results indicate that ING1b epigenetically regulates several miRNAs including miR-203. The several-fold increase in miR-203 by ING1b might inhibit cancer cell proliferation through coordinate downregulation of CDK6, c-Abl and Src.

// Xiaolan Feng 1,2,3 , Haocheng Li 3,4 , Elizabeth N. Kornaga 5,6 , Michelle Dean 5,6 , Susan P. Lees-Miller 7 , Karl Riabowol 7 , Anthony M. Magliocco 8 , Don Morris 3,6 , Peter H. Watson 9 , Emeka K Enwere 5,6 , Gwyn Bebb 3,6 and Alexander Paterson 3 1 Department of Oncology, BC Cancer Agency-Vancouver Island Center, Victoria, British Columbia, Canada 2 Faculty of Medicine, The University of British Columbia, Vancouver, British Columbia, Canada 3 Department of Oncology, Tom Baker Cancer Centre and University of Calgary, Cumming School of Medicine, Calgary, Alberta, Canada 4 Department of Community Health Science, TRW Building, University of Calgary, Calgary, Alberta, Canada 5 Functional Tissue Imaging Unit, Translational Research Laboratory, Tom Baker Cancer Centre, Calgary, Alberta, Canada 6 Translational Research Laboratory, Tom Baker Cancer Centre, Calgary, Alberta, Canada 7 Department of Biochemistry and Molecular Biology, Health Science Building, University of Calgary, Alberta, Canada 8 Department of Anatomic Pathology, H. Lee Moffitt Cancer Center, Tampa, FL, USA 9 Department of Pathology, BC Cancer Agency-Vancouver Island Center, Victoria, British Columbia, Canada Correspondence to: Xiaolan Feng, email: // Keywords : ATM, Ki67, early stage hormone receptor positive breast cancer, automated quantitative immunofluorescence analysis, disease specific overall survival Received : August 26, 2016 Accepted : October 05, 2016 Published : October 12, 2016 Abstract Introduction: This study was designed to investigate the combined influence of ATM and Ki67 on clinical outcome in early stage hormone receptor positive breast cancer (ES-HPBC), particularly in patients with smaller tumors (< 4 cm) and fewer than four positive lymph nodes. Methods: 532 formalin-fixed paraffin-embedded specimens of resected primary breast tumors were used to construct a tissue microarray. Samples from 297 patients were suitable for final statistical analysis. We detected ATM and Ki67 proteins using fluorescence and brightfield immunohistochemistry respectively, and quantified their expression with digital image analysis. Data on expression levels were subsequently correlated with clinical outcome. Results: Remarkably, ATM expression was useful to stratify the low Ki67 group into subgroups with better or poorer prognosis. Specifically, in the low Ki67 subgroup defined as having smaller tumors and no positive nodes, patients with high ATM expression showed better outcome than those with low ATM, with estimated survival rates of 96% and 89% respectively at 15 years follow up ( p = 0.04). Similarly, low-Ki67 patients with smaller tumors, 1-3 positive nodes and high ATM also had significantly better outcomes than their low ATM counterparts, with estimated survival rates of 88% and 46% respectively ( p = 0.03) at 15 years follow up. Multivariable analysis indicated that the combination of high ATM and low Ki67 is prognostic of improved survival, independent of tumor size, grade, and lymph node status ( p = 0.02). Conclusions: These data suggest that the prognostic value of Ki67 can be improved by analyzing ATM expression in ES-HPBC.

Epigenetic, transcriptional and signaling processes in the nucleolus regulate rRNA transcription and cell growth. We report here that the tumor suppressor ING1b binds rDNA, regulates rDNA chromatin modifications and affects nucleolar localization of mTOR to modulate rRNA levels. ING1 represses rDNA transcription by recruiting HDAC1 to rDNA loci, increasing its association with the NoRC complex and deacetylating the histone H3K9 and H3K27 marks of active transcription. Loss of ING1 enhances nucleolar localization of phospho-mTOR and its association with Raptor and GβL, even during rapamycin treatment. ING1 inhibits rDNA transcription by inhibiting UBF activity and its interaction with mTOR. Regulation of rDNA heterochromatin and rRNA synthesis by ING1 is also apparent during normal cell growth and during cell stress. Moreover, this function was also important during PMA induced differentiation of THP1 cells, since knocking down ING1 affected the process by inhibiting rRNA transcriptional repression. These observations show that ING1 regulates the nucleolar epigenome and rDNA transcription suggesting that regulation of protein synthesis might serve as the basis for ING1 function as a type II tumor suppressor.

Background: Breast cancer accounts for around 45,000 cancer related deaths in North America per year. The high incidence of breast cancer observed in this region is most likely due to the availability of various screening programs used to detect breast cancer, which otherwise may never get diagnosed. There is an inverse relation between the cost of treatment and patient survival as the breast cancer progresses to higher grades. Therefore, screening and diagnosing breast cancers at earlier stages is needed which can improve patient survival. The INhibitor of Growth (ING) family of tumour suppressor proteins are implicated in diverse cellular processes including chromatin remodelling and apoptosis. ING proteins are stoichiometric members of histone acetyl transferase (HAT) and histone deacetylase (HDAC) complexes. Their expression is downregulated in several cancers, including breast carcinoma. Alternatively, ING function may be compromised by mislocalization to the cytoplasm. However, the association of expression of ING proteins and cancer specific survival has not been studied yet. In this study, we aimed to evaluate the prognostic significance of ING1 in breast cancer. Patients and Methods: This study included 443 Breast Cancer patients diagnosed in Calgary, Canada from 1985–2000. Clinical data were obtained from the Alberta Cancer Registry and chart review. Tissue microarrays (TMAs) were assembled from triplicate cores of formalin-fixed paraffin-embedded tumour tissue. ING1 protein expression was quantified using fluorescent immunohistochemistry and automated quantitative analysis (AQUA) in normal breast epithelial cells and breast cancer samples. 5 year progression free survival (PFS) was analyzed using Kaplan-Meier plots and Cox proportional hazard modelling. Results: In our study, we found that ING1 expression is significantly downregulated in the breast cancer patient samples relative to normal breast epithelia. High or low ING1 cytoplasmic/nuclear (C/N) ratio results in poor 5 year PFS. Also, the low tumor/stromal (t/s) ratio of ING1 results in poor prognosis. In univariate analysis, t/s ING1 expression showed a negative correlation with tumor stage [p = 0.008]; tumor grade [p = 0.000]; nodal status [p = 0.000] and 5 year PFS [p = 0.004]. However, no correlation was observed with ER, PR or HER2 status. In multivariate analysis, t/s ING1 proved to be an independent and better prognostic marker [HR 0.582, p = 0.037] than HER2 [HR 2.230, p = 0.057]; Age at diagnosis [HR 2.010, p = 0.205] and tumor size [HR 1.471, p = 0.189]. Discussion and Conclusions: This is the first study to evaluate ING1 expression in breast cancer using the AQUA technique. ING1 expression high or low in the C/N ratio might disrupt the stoichiometry of chromatin-modifying complexes and hamper ING1s extra nuclear functions, leading to poor survival. The significantly improved survival observed in patients with high t/s ING1 expression further strengthens the role of ING1 as a tumor suppressor. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P4-09-07.

Lung cancer is the leading cause of cancer death in the U.S. and remains the least-funded cancer in North America. Many studies addressed disparities in lung cancer incidence rate among the races; however, few studies described the effect of both race and gender in the risk of manifesting this disease. These studies lack consensus for both the etiology and the magnitude of gender-based disparities and their impact on disease development on specific racial groups. These unknown race-associated characteristics between men and women might adversely affect the improvement of current therapies and patient outcomes. Characterizing gender as a possible risk modifier in lung cancer development among certain ethnic groups may aid in the development of targeted agents that improve patient outcomes. This study aimed to investigate the global pattern of gender-based disparities in incidence of lung cancer and describe the etiologic factors associated with different racial groups in non-small cell lung cancer (NSCLC), since it comprises 85% of all diagnosed cases. This aim was accomplished by analyzing data scrutinized using a systematic review approach for studies published between 1996 and 2016. We found a statistically significant effect of race on lung cancer incidence rate, and this effect varies by gender. We also found that certain races are more prone to develop adenocarcinoma in their histology than others, regardless of their sex. In addition, we found that Asian women have higher rates of NSCLC regardless of their living environment. Our findings also show that the number of cases of NSCLC is rising among women of all races, regardless of their smoking status. By visualizing NSCLC pattern in both men and women among different countries and racial groups, we stimulate future findings to establish efficacious preventative strategies and will stimulate research advancements toward sex- and racial-designed diagnostics, including alternative treatments that could reduce the manifestation of this disease. Note: This abstract was not presented at the conference. Citation Format: Noor Alsaadoun, Karen Kopciuk, Desiree Hao, Karl Riabowol, D. Morley Hollenberg, D. Gwyn Bebb. Gender and racial disparities in non-small cell lung cancer: A systematic review [abstract]. In: Proceedings of the Tenth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2017 Sep 25-28; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2018;27(7 Suppl):Abstract nr A89.

Replication-competent oncolytic viruses hold great potential for the clinical treatment of many cancers. Importantly, many oncolytic virus candidates, such as reovirus and myxoma virus, preferentially infect cancer cells bearing abnormal cellular signaling pathways. Reovirus and myxoma virus are highly responsive to activated Ras and Akt signaling pathways, respectively, for their specificity for viral oncolysis. However, considering the complexity of cancer cell populations, it is possible that other tumor-specific signaling pathways may also contribute to viral discrimination between normal versus cancer cells. Because carcinogenesis is a multistep process involving the accumulation of both oncogene activations and the inactivation of tumor suppressor genes, we speculated that not only oncogenes but also tumor suppressor genes may have an important role in determining the tropism of these viruses for cancer cells. It has been previously shown that many cellular tumor suppressor genes, such as p53, ATM and Rb, are important for maintaining genomic stability; dysfunction of these tumor suppressors may disrupt intact cellular antiviral activity due to the accumulation of genomic instability or due to interference with apoptotic signaling. Therefore, we speculated that cells with dysfunctional tumor suppressors may display enhanced susceptibility to challenge with these oncolytic viruses, as previously seen with adenovirus. We report here that both reovirus and myxoma virus preferentially infect cancer cells bearing dysfunctional or deleted p53, ATM and Rb tumor suppressor genes compared to cells retaining normal counterparts of these genes. Thus, oncolysis by these viruses may be influenced by both oncogenic activation and tumor suppressor status.

Summary The ING family of tumor suppressor proteins affects cell growth, apoptosis and response to DNA damage by modulating chromatin structure through association with different HAT and HDAC complexes. The major splicing isoforms of the ING1 locus are ING1a and INGlb. While INGlb plays a role in inducing apoptosis, the function of ING1a is currently unknown. Here we show that alternative splicing of the ING1 message alters the INGla:INGlb ratio by ~30-fold in senescent compared to low passage primary fibroblasts. INGla antagonizes INGlb function in apoptosis, induces the formation of structures resembling senescence- associated heterochromatic foci containing heterochromatin protein 1 gamma, the accumulation of senescence-associated β β β -galactosidase activity and promotes senescent cell mor- phology and cell cycle arrest. Phenotypic effects may result from differential effects on gene expression since ING1a increases levels of both retinoblastoma and the p16 cyclin-dependent kinase inhibitor and ING1a and ING1b have opposite effects on the expression of proliferating nuclear cell antigen (PCNA), which is required for cell growth. Gene expression appears to be altered by targeting of HDAC complexes to gene promoters since INGla associates with several-fold higher levels of HDAC1 in senescent, compared to replication-competent cells and ING1 is found on the PCNA promoter by chromatin immuno- precipitation analysis. These data demonstrate a novel role for the ING1 proteins in differentially regulating senescence-associated chromatin remodeling vs. apoptosis and support the idea that altered ratios of the ING1 splicing isoforms may contribute to establishing the senescent phenotype through HDAC and HAT complex-mediated effects on chromatin structure.

The INhibitor of Growth (ING) family of type II tumour suppressors are encoded by five genes in mammals (ING1-ING5), most of which encode multiple isoforms via splicing, and all of which contain a highly conserved plant homeodomain (PHD) finger motif. Since their discovery approximately ten years ago, significant progress has been made in understanding their subcellular targeting, their relationship to p53, their activation by bioactive phospholipids, and their key role in reading the histone code via PHD fingers, with subsequent effects on histone acetylation and transcriptional regulation. In the past year, we have begun to understand how ING proteins integrate stress signals with interpretation and modification of the histone epigenetic code to function as tumour suppressors.

The efficacy of most cancer treatments depends markedly on the high replication rate of cancer cells, a characteristic frequently observed in neoplasms with higher grades of malignancy. Yet, the same characteristic is present in many normal regenerative tissues of the body, which makes them susceptible to the cytotoxic effects of chemotherapeutics and accounts for many of the toxic side effects of these drugs. In response to cell killing by chemotherapeutics, normal regenerative tissues replicate at a faster rate to regenerate, resulting in accelerated telomere attrition and leaving different cell populations with telomeres shorter than they would normally have in the absence of treatment. This accelerated erosion has implications regarding the recurrence of cancers at secondary sites because reduced replicative ability may compromise effective subsequent immune responses. In this review we discuss recent reports describing the effect of chemotherapeutics on telomere loss, how this may impact healthy tissues in an age-dependent manner, and describe in brief emerging cancer treatments that may avoid this telomere erosion effect.

Impaired tumor suppressor functions, such as deficient p53, are characteristic for glioblastoma multiforme (GBM) and can cause resistance to DNA-damaging agents like cisplatin. We have recently shown that the INhibitor of Growth 1 (ING1) tumor suppressor is down-regulated in malignant gliomas and that the decrease of ING1 expression correlates with histological grade of malignancy, suggesting a role for ING1 in the pathogenesis and progression of malignant gliomas. Based on this background, the purpose of our current study was to examine the potential impact of ING1 protein levels on DNA-damage response in GBM. Using LN229 GBM cells, which express ING1 proteins and harbor mutant TP53, we are the first to show that DNA damage by cisplatin or ionizing radiation differentially induced the two major ING1 splicing isoforms. The p47ING1a isoform, that promotes deacetylation of histones, thus formation of heterochromatic regions of DNA, which are less susceptible to DNA damage, was preferentially induced by >50-fold. This might represent a response to protect DNA from damage. Also, ING1 knockdown by siRNA accelerated transit of cells through G1 phase, consistent with ING1 serving a tumor suppressor function, and caused cells to enter apoptosis more rapidly in response to cisplatin. Our results indicate that malignant gliomas may down-regulate ING1 to allow more efficient tumor growth and progression. Also, ING1 down-regulation may sensitize GBM cells with deficient p53 to treatment with cisplatin.

The inhibitor of growth family member 3 (ING3) is a member of the ING tumor suppressor family. Although its expression has been reported in various types of cancers, the role of ING3 and its prognostic value in prostate cancer (PCa) has not been investigated. ING3 expression and prognostic value was assessed in a cohort of PCa patients (n = 312) treated with transurethral resection of prostate using immumoflourescent automated quantitative analysis (AQUA) system. In vitro studies were carried out in conjunction to investigate its expression in various PCa cell lines. ING3 knockdown was also carried out in DU145 cell lines to assess for any changes in invasion and migration. ING3 expression was highest in benign prostate tissues (mean 3.2 ± 0.54) compared to PCa (mean 2.5 ± 0.26) (p = 0.437), advanced prostate cancer (AdvPCa) (mean 1.5 ± 0.32) (p = 0.004), and castration-resistant prostate cancer (CRPC) (mean 2.28 ± 0.32) (p = 0.285). ING3 expression was inversely correlated to Gleason score (p = 0.039) and ETS-related gene (ERG) expression (p = 0.019). Higher ING3 expression was marginally associated with lethal disease (p = 0.052), and this was more pronounced in patients with ERG-negative status (p = 0.018). Inhibition of ING3 in DU145 PCa cells using small interfering RNA (siRNA) was associated with decreased cell invasion (p = 0.0016) and cell migration compared to control cells. ING3 is significantly associated with PCa disease progression and cancer-specific mortality. To our knowledge, this is the first report suggesting an oncogenic function of ING3, previously well known as a tumor suppressor protein. Further studies should investigate potential-related pathways in association to ING3.

This protocol presents a rapid, efficient, and practical (REAP) method to separate nuclei from cultured cells in vitro with as little damage and contamination as possible. The REAP procedure is performed at low temperature and takes

The isolation of nuclei is often the first step in studying processes such as nuclear-cytoplasmic shuttling, subcellular localization of proteins, and protein–chromatin or nuclear protein–protein interactions in response to diverse stimuli. Therefore, rapidly obtaining nuclei from cells with relatively high purity and minimal subcellular contamination, protein degradation, or postharvesting modification is highly desirable. Historically, the isolation of nuclei involved a homogenization step followed by centrifugation through high-density glycerol or sucrose. Although clean nuclei with little cytoplasmic contamination can be prepared using this method, it is typically time consuming and can allow protein degradation, protein modification, and leaching of components from the nuclei to occur. We have developed a rapid and simple fractionation method that is based on the selective dissolution of the cytoplasmic membrane (but not the nuclear membrane) using a low concentration of a nonionic detergent and rapid centrifugation steps. Here we describe important considerations when isolating nuclei from cells, introduce our rapid method, and compare this method to a more traditional protocol for isolating nuclei, noting the strengths and limitations of each approach.

Background Previous studies have established that levels of the Inhibitor of Growth 1(ING1) tumor suppressor are reduced in a significant proportion of different cancer types. Here we analyzed levels of ING1 in breast cancer patients to determine its prognostic significance as a biomarker for breast cancer prognosis. Methods We used automated quantitative analysis (AQUA) to determine the levels of ING1 in the tumor associated stromal cells of 462 breast cancer samples. To better understand how high ING1 levels affect nearby epithelium, we measured the levels of cytokines and secreted matrix metalloproteases (MMPs), using an ELISA based assay in mammary fibroblasts overexpressing ING1. These cells were also used in a 3-dimensional co-culture with MCF7 cells to determine the effect of released MMPs and other cytokines on growing colonies. Results We find that high levels of ING1 in stroma are associated with tumor grade (p = 0.001) and size (p = 0.02), and inversely associated with patient survival (p = 0.0001) in luminal, but not in non-luminal cancers, suggesting that high stromal ING1 promotes cancer development. In this group of patients ING1 could also predict patient survival and act as a biomarker (HR = 2.125). While ING1 increased or decreased the expression of different cytokines, ING1 also increased the levels of MMP1, MMP3 and MMP10 by 5–8 fold, and concomitantly decreased levels of the tissue inhibitors of metalloproteases TIMP2, TIMP3 and TIMP4 by 1.5–3.3 fold, resulting in significant increases in MMP activity as determined by zymography. Co-culturing of MCF7 cells with stromal cells expressing ING1 in 3-dimensional organoid cultures suggested that MCF7 colonies were less well defined, suggesting that secreted MMPs might promote migration. Conclusion These data indicate that stromal ING1 expression can predict the survival of patients with luminal breast cancer. High levels of ING1 in stromal cells can promote the development of breast cancer through increased expression and release of MMPs and down regulation of TIMPs, which may be an underlying mechanism of reduced patient survival. Electronic supplementary material The online version of this article (doi:10.1186/s12943-015-0434-x) contains supplementary material, which is available to authorized users.

ING proteins affect apoptosis, growth, and DNA repair by transducing stress signals such as DNA damage, binding histones, and subsequently regulating chromatin structure and p53 activity. p53 target genes, including the p21 cyclin-dependent kinase inhibitor and Bax, an inducer of apoptosis, are regulated by ING proteins. To identify additional targets downstream of p33ING1 and p32ING2, cDNA microarrays were performed on phenotypically normal human primary fibroblasts. The 0.36% of genes affected by ING proteins in primary fibroblasts were distinct from targets seen in established cells and included the HSP70 heat shock gene, whose promoter was specifically induced10-fold. ING1-induced expression of HSP70 shifted cells from survival to a death pathway in response to tumor necrosis factor alpha (TNF-alpha), and p33ING1b protein showed synergy with TNF-alpha in inducing apoptosis, which correlated with reduced NF-kappaB-dependent transcription. These findings are consistent with previous reports that HSP70 promotes TNF-alpha-mediated apoptosis by binding I-kappaBeta kinase gamma and impairing NF-kappaB survival signaling. Induction of HSP70 required the amino terminus of ING1b but not the plant homeodomain region that was recently identified as a histone binding domain. Regulation of HSP70 gene expression by the ING tumor suppressors provides a novel link between the INGs and the stress-regulated NF-kappaB survival pathway important in hypoxia and angiogenesis.

The INhibitor of Growth (ING) family of plant homeodomain (PHD) proteins induce apoptosis and regulate gene expression through stress-inducible binding of phospholipids with subsequent nuclear and nucleolar localization. Relocalization occurs concomitantly with interaction with a subset of nuclear proteins, including PCNA, p53 and several regulators of acetylation such as the p300/CBP and PCAF histone acetyltransferases (HATs), as well as the histone deacetylases HDAC1 and hSir2. These interactions alter the localized state of chromatin compaction, subsequently affecting the expression of subsets of genes, including those associated with the stress response (Hsp70), apoptosis (Bax, MDM2) and cell cycle regulation (p21WAF1, cyclin B) in a cell- and tissue-specific manner. The expression levels and subcellular localization of ING proteins are altered in a significant number of human cancer types, while the expression of ING isoforms changes during cellular aging, suggesting that ING proteins may play a role in linking cellular transformation and replicative senescence. The variety of functions attributed to ING proteins suggest that this tumor suppressor serves to link the disparate processes of cell cycle regulation, cell suicide and cellular aging through epigenetic regulation of gene expression. This review examines recent findings in the ING field with a focus on the functions of protein-protein interactions involving ING family members and the mechanisms by which these interactions facilitate the various roles that ING proteins play in tumorigenesis, apoptosis and senescence.

ING1 is a type II tumor suppressor that affects cell growth, stress signaling, apoptosis, and DNA repair by altering chromatin structure and regulating transcription. Decreased ING1 expression is seen in several human cancers, and mislocalization has been noted in diverse types of cancer cells. Aberrant targeting may, therefore, functionally inactivate ING1. Bioinformatics analysis identified a sequence between the nuclear localization sequence and plant homeodomain domains of ING1 that closely matched the binding motif of 14-3-3 proteins that target cargo proteins to specific subcellular locales. We find that the widely expressed p33(ING1b) splicing isoform of ING1 interacts with members of the 14-3-3 family of proteins and that this interaction is regulated by the phosphorylation status of ING1. 14-3-3 binding resulted in significant amounts of p33(ING1b) protein being tethered in the cytoplasm. As shown previously, ectopic expression of p33(ING1b) increased levels of the p21(Waf1) cyclin-dependent kinase inhibitor upon UV-induced DNA damage. Overexpression of 14-3-3 inhibited the up-regulation of p21(Waf1) by p33(ING1b), consistent with the idea that mislocalization blocks at least one of ING1's biological activities. These data support the idea that the 14-3-3 proteins play a crucial role in regulating the activity of p33(ING1b) by directing its subcellular localization.

Senescence was originally defined as a state associated with cell cycle arrest that occurs after cells have undergone an intrinsically limited number of divisions in vitro. Much evidence indicates that senescence occurs as a consequence of the internal stress signal generated from shortening telomeres on the ends of chromosomes. However, more recently, various forms of extrinsic stresses have been shown to induce a markedly similar senescent phenotype that includes changes in chromatin structure and gene expression. Chromatin structure is subject to many forms of modification that affect transcription, gene silencing, cell proliferation, and senescence, much of which involves imposition of an epigenetic histone code. Several genes in the p53, Rb, and ING (inhibitor of growth) pathways affect cell senescence and are capable of regulating gene expression through chromatin remodeling. This suggests that a link may exist between chromatin modification and cellular senescence through the activity of proteins typically defined as tumor suppressors.

Telomere length is linked to age-associated diseases, with shorter telomeres in blood associated with an increased probability of mortality from infection or heart disease. Little is known about how human telomere length is regulated despite convincing data from twins that telomere length is largely heritable, uniform in various tissues during development until birth and variable between individuals. As sperm cells show increasing telomere length with age, we investigated whether age of fathers at conception correlated with telomere length of their offspring. Telomere length in blood from 125 random subjects was shown to be positively associated with paternal age (+22 bp yr -1, 95% confidence interval 5.2-38.3, P = 0.010), and paternal age was calculated to affect telomere length by up to 20% of average telomere length per generation. Males lose telomeric sequence faster than females (31 bp yr -1, 17.6-43.8, P < 0.0001 vs. 14 bp yr -1, 3.5-24.8, P < 0.01) and the rate of telomere loss slows throughout the human lifespan. These data indicate that paternal age plays a role in the vertical transmission of telomere length and may contribute significantly to the variability of telomere length seen in the human population, particularly if effects are cumulative through generations.

Fibroblasts lose the ability to replicate in response to growth factors and become unable to express growth-associated immediate-early genes, including c-fos and egr-1, as they become senescent. The serum response factor (SRF), a major transcriptional activator of immediate-early gene promoters, loses the ability to bind to the serum response element (SRE) and becomes hyperphosphorylated in senescent cells. We identify protein kinase C delta (PKC delta) as the kinase responsible for inactivation of SRF both in vitro and endogenously in senescent cells. This is due to a higher level of PKC delta activity as cells age, production of the PKC delta catalytic fragment, and its nuclear localization in senescent but not in low-passage-number cells. The phosphorylation of T160 of SRF by PKC delta in vitro and in vivo led to loss of SRF DNA binding activity. Both the PKC delta inhibitor rottlerin and ectopic expression of a dominant negative form of PKC delta independently restored SRE-dependent transcription and immediate-early gene expression in senescent cells. Modulation of PKC delta activity in vivo with rottlerin or bistratene A altered senescent- and young-cell morphology, respectively. These observations support the idea that the coordinate transcriptional inhibition of several growth-associated genes by PKC delta contributes to the senescent phenotype.

The ING family of proteins are involved in chromatin remodelling, and bind to and affect the activity of histone acetyltransferase, histone deacetylase, and factor acetyltransferase protein complexes. Some family members affect transcription, including the expression of p53-inducible genes such as p21 and Bax, and ING2 induces p53 acetylation on a site implicated in the regulation of p53 activity. ING1 promotes DNA repair and interacts with proliferating cell nuclear antigen, thus linking DNA repair, apoptosis and chromatin remodelling. Here, we summarize what is known about the molecular interactions of ING1 family proteins and, based on these interactions, develop a model to better understand the impact of ING proteins on multiple biological processes.