ING1 Protein Research Articles

Erratum: ING1 induces apoptosis through direct effects at the mitochondria

The ING family of tumor suppressors acts as readers and writers of the histone epigenetic code, affecting DNA repair, chromatin remodeling, cellular senescence, cell cycle regulation and apoptosis. The best characterized member of the ING family, ING1, interacts with the proliferating cell nuclear antigen (PCNA) in a UV-inducible manner. ING1 also interacts with members of the 14-3-3 family leading to its cytoplasmic relocalization. Overexpression of ING1 enhances expression of the Bax gene and was reported to alter mitochondrial membrane potential in a p53-dependent manner. Here we show that ING1 translocates to the mitochondria of primary fibroblasts and established epithelial cell lines in response to apoptosis inducing stimuli, independent of the cellular p53 status. The ability of ING1 to induce apoptosis in various breast cancer cell lines correlates well with its degree of translocation to the mitochondria after UV treatment. Endogenous ING1 protein specifically interacts with the pro-apoptotic BCL2 family member BAX, and colocalizes with BAX in a UV-inducible manner. Ectopic expression of a mitochondria-targeted ING1 construct is more proficient in inducing apoptosis than the wild type ING1 protein. Bioinformatic analysis of the yeast interactome indicates that yeast ING proteins interact with 64 mitochondrial proteins. Also, sequence analysis of ING1 reveals the presence of a BH3-like domain. These data suggest a model in which stress-induced cytoplasmic relocalization of ING1 by 14-3-3 induces ING1-BAX interaction to promote mitochondrial membrane permeability and represent a paradigm shift in our understanding of ING1 function in the cytoplasm and its contribution to apoptosis.

ING1 induces apoptosis through direct effects at the mitochondria

The ING family of tumor suppressors acts as readers and writers of the histone epigenetic code, affecting DNA repair, chromatin remodeling, cellular senescence, cell cycle regulation and apoptosis. The best characterized member of the ING family, ING1, interacts with the proliferating cell nuclear antigen (PCNA) in a UV-inducible manner. ING1 also interacts with members of the 14-3-3 family leading to its cytoplasmic relocalization. Overexpression of ING1 enhances expression of the Bax gene and was reported to alter mitochondrial membrane potential in a p53-dependent manner. Here we show that ING1 translocates to the mitochondria of primary fibroblasts and established epithelial cell lines in response to apoptosis inducing stimuli, independent of the cellular p53 status. The ability of ING1 to induce apoptosis in various breast cancer cell lines correlates well with its degree of translocation to the mitochondria after UV treatment. Endogenous ING1 protein specifically interacts with the pro-apoptotic BCL2 family member BAX, and colocalizes with BAX in a UV-inducible manner. Ectopic expression of a mitochondria-targeted ING1 construct is more proficient in inducing apoptosis than the wild type ING1 protein. Bioinformatic analysis of the yeast interactome indicates that yeast ING proteins interact with 64 mitochondrial proteins. Also, sequence analysis of ING1 reveals the presence of a BH3-like domain. These data suggest a model in which stress-induced cytoplasmic relocalization of ING1 by 14-3-3 induces ING1-BAX interaction to promote mitochondrial membrane permeability and represent a paradigm shift in our understanding of ING1 function in the cytoplasm and its contribution to apoptosis.

ING1b-inducible microRNA203 inhibits cell proliferation

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.

Abstract P4-09-07: ING1 Expression Measured by AQUA can be an Independent Prognostic Marker in Breast Cancer

Abstract 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.

Human Family with Sequence Similarity 60 Member A (FAM60A) Protein: a New Subunit of the Sin3 Deacetylase Complex

Here we describe the function of a previously uncharacterized protein, named family with sequence similarity 60 member A (FAM60A) that maps to chromosome 12p11 in humans. We use quantitative proteomics to determine that the main biochemical partners of FAM60A are subunits of the Sin3 deacetylase complex and show that FAM60A resides in active HDAC complexes. In addition, we conduct gene expression pathway analysis and find that FAM60A regulates expression of genes that encode components of the TGF-beta signaling pathway. Moreover, our studies reveal that loss of FAM60A or another component of the Sin3 complex, SDS3, leads to a change in cell morphology and an increase in cell migration. These studies reveal the function of a previously uncharacterized protein and implicate the Sin3 complex in suppressing cell migration.

Expression and Analysis of P33/ING1 and HIF-1α in Invasive Ductal Carcinoma of Breast

Objective To study the expression and significance of P33/ING1 and HIF-1α in invasive ductal carcinoma of breast(IDC).Methods Immunohistochemical SP method was performed to detect the expression of P33/ING1 and HIF-1α in 68 cases of IDC and 20 cases of breast fibroadenoma and the relationship between the expression and clinical-pathological characteristics was analyzed.Results The positive expression rates of P33/ING1 protein in IDC was significant lower than that in breast fibroadenoma(60.29% vs.95%;P0.01);and negatively correlated with tumor size,axillary lymph node metastasis and histologic grade(P0.05).The positive expression rates of HIF-1α protein in IDC was significant higher than that in breast fibroadenoma(52.94% vs.10%;P0.01) and positively correlated with axillary lymph node metastasis and histologic grade(P0.05).There was a negatively correlation between P33/ING1 and HIF-1α in IDC(r=-0.283;P0.05).Conclusion There were low-expression of P33/ING1 and over-expression of HIF-1α in IDC.P33/ING1 and HIF-1α proteins were significantly correlated with the clinicopathological characteristics in IDC.Combined detection of P33/ING1 and HIF-1α might calculate the biological behaviors of IDC.

The tumor suppressor p33ING1b upregulates p16INK4a expression and induces cellular senescence

ING1 protein is a tumor suppressor which plays significant roles in multiple cellular activities. p47ING1a and p33ING1b are major splice isoforms of ING1 and their roles in senescence need further investigations. Here we studied the functions of ING1 isoforms in cellular senescence and gene regulation, with focus on p16INK4a. We observe that p33ING1b protein is the major ING1 isoform expressed in 2BS human diploid fibroblasts. Overexpression of p33ING1b induces cellular senescence and upregulates p16INK4a expression in 2BS fibroblasts. p33ING1b upregulates p16INK4a transcription. p33ING1b and p300 bind to the p16INK4a promoter. p300/CBP-specific inhibitor curcumin can reverse the induction of p16INK4a by p33ING1b. These results help to better understand the function of ING1.

The ING tumor suppressors in cellular senescence and chromatin

The Inhibitor of Growth (ING) proteins represent a type II tumor suppressor family comprising five conserved genes, ING1 to ING5. While ING1, ING2 and ING3 proteins are stable components of the mSIN3a-HDAC complexes, the association of ING1, ING4 and ING5 with HAT protein complexes was also reported. Among these the ING1 and ING2 have been analyzed more deeply. Similar to other tumor suppressor factors the ING proteins are also involved in many cellular pathways linked to cancer and cell proliferation such as cell cycle regulation, cellular senescence, DNA repair, apoptosis, inhibition of angiogenesis and modulation of chromatin.A common structural feature of ING factors is the conserved plant homeodomain (PHD), which can bind directly to the histone mark trimethylated lysine of histone H3 (H3K4me3). PHD mutants lose the ability to undergo cellular senescence linking chromatin mark recognition with cellular senescence. ING1 and ING2 are localized in the cell nucleus and associated with chromatin modifying enzymes, linking tumor suppression directly to chromatin regulation. In line with this, the expression of ING1 in tumors is aberrant or identified point mutations are mostly localized in the PHD finger and affect histone binding. Interestingly, ING1 protein levels increase in replicative senescent cells, latter representing an efficient pathway to inhibit cancer proliferation. In association with this, suppression of p33ING1 expression prolongs replicative life span and is also sufficient to bypass oncogene-induced senescence. Recent analyses of ING1- and ING2-deficient mice confirm a tumor suppressive role of ING1 and ING2 and also indicate an essential role of ING2 in meiosis.Here we summarize the activity of ING1 and ING2 as tumor suppressors, chromatin factors and in development.

ING1a expression increases during replicative senescence and induces a senescent phenotype

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 approximately 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 beta-galactosidase activity and promotes senescent cell morphology 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 immunoprecipitation 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.

ING1 protein targeting to the nucleus by karyopherins is necessary for activation of p21

ING1 proteins affect apoptosis, growth, and DNA repair by binding histones and regulating chromatin structure and gene expression. ING1 is downregulated in cancers and cytoplasmic localization is associated with poor prognosis. Here, we report that ING1b interacts with karyopherins α2 and β1 through several basic nuclear localization sequences (NLS) located adjacent to the ING1b PHD region. Deletion of NLS motifs resulted in failure of ING1b to completely localize to the nucleus and inhibited its ability to induce p21WAF1 expression. These observations support a general mechanism by which ING1b activity is regulated, in part, through dynamic subcellular partitioning between the nucleus and cytoplasm.

No ING1 mutations in human brain tumours but reduced expression in high malignancy grades of astrocytoma.

The ING1 family of proteins has been shown to have regulatory functions in oncogenesis, apoptosis, DNA repair and cell cycle regulation. Here we present the first report on LOH analysis of the ING1 locus, mutation analysis of the complete coding sequence including intron-exon boundaries and expression analysis of the different ING1 splice products and protein isoforms in primary brain tumours. No somatic ING1 mutations were detected. Semi-quantitative analysis revealed higher levels of p33ING1b RNA in benign than in malignant lesions. This correlation was significant in a subset of 37 astrocytic tumours WHO grades I to IV. ING1 protein isoforms p47ING1a, p33ING1b and p24ING1c were found to be expressed variably in this series. Our findings support a regulatory contribution of ING1 to the development or progression of brain tumours.

P33ING1b and estrogen receptor (ER) alpha.

The ING1 gene was originally cloned as a candidate tumor suppressor of human breast cancer, and recent studies suggest that ING1 proteins are involved in chromatin remodeling functions via physical association with both histone acetyltransferases (HATs) and histone deacetylases (HDACs). Both CREB binding protein (CBP) and the related p300 proteins show a marked preference for binding to complexes containing p33ING1b, one of the major ING1 isoforms, whereas HDAC immunocomplexes contain equal amounts of p33ING1b and p47ING1a. This observation is interesting, given that p33ING1b can selectively increase histone H3 and H4 acetylation when micro-injected into individual cells, whereas p47ING1a inhibits histone acetylation. We investigated whether p33ING1b modulated the transcriptional activity of estrogen receptor (ER)alpha. In cells transfected with increasing concentrations of a mammalian expression vector encoding p33ING1b, estrogen-induced ER alpha transcriptional activity was found to increase in a dose-dependent manner. As p33ING1b expression levels increased, transcription of an ER-responsive reporter gene by either estrogen-inducible full-length ER alpha or the activation function (AF) 1 deletion mutant was enhanced, while the AF2 deletion mutant was unaffected by the presence of p33ING1b. These results showed that p33ING1b enhanced estrogen-induced ER alpha activity through the AF2 domain. Our data demonstrate that p33ING1b acts like a coactivator for ER alpha and stimulates estrogen-induced ER alpha transcriptional activity consistent with a function for p33ING1b in chromatin remodeling.

P33 ING1b stimulates the transcriptional activity of the estrogen receptor α via its activation function (AF) 2 domain

The ING1 gene was originally cloned as a candidate tumor suppressor of human breast cancer, and recent studies suggest that ING1 proteins are involved in chromatin remodeling functions via physical association with both histone acetyltransferases (HATs) and histone deacetylases (HDACs). In this study, we investigated whether p33 ING1b, one of the major ING1 isoforms, modulated the transcriptional activity of estrogen receptor (ER) α. In Cos-7 cells transfected with increasing concentrations of a mammalian expression vector encoding for p33 ING1b, estrogen-induced ERα transcriptional activity was found to increase in a dose-dependent manner. As p33 ING1b expression levels increased, transcription of an ER-responsive reporter gene by either estrogen-inducible full-length ERα or activation function (AF) 1 deletion mutant was enhanced, while the AF2 deletion mutant was unaffected by the presence of p33 ING1b. These results showed that p33 ING1b enhanced estrogen-induced ERα activity through the AF2 domain. Our data also demonstrated that the antiestrogens inhibited the transcriptional activity of ERα as stimulated by p33 ING1b. Furthermore, a weak physical association was observed between in vitro translated p33 ING1b and ERα. Our data presented here demonstrate that p33 ING1b acts like a coactivator for ERα and stimulates estrogen-induced ERα transcriptional activity consistent with a function for p33 ING1b in chromatin remodeling.

P33 ING1 Enhances UVB-Induced Apoptosis in Melanoma Cells

The biological functions of the tumor suppressor ING1 have been studied extensively in the past few years since it was cloned. It shares many biological functions with p53 and has been reported to mediate growth arrest, senescence, apoptosis, anchorage-dependent growth, chemosensitivity, and DNA repair. Some of these functions, such as cell cycle arrest and apoptosis, have been shown to be dependent on the activity of both ING1 and p53 proteins. Two recent reports by Scott and colleagues demonstrate that p33 ING1 (one of the ING1 isoforms) translocates to the nucleus and binds to PCNA upon UV irradiation. Here we report that p33 ING1 mediates UV-induced cell death in melanoma cells. We found that overexpression of p33 ING1 increased while the introduction of an antisense p33 ING1 plasmid reduced the apoptosis rate in melanoma cells after UVB irradiation. We also demonstrated that enhancement of UV-induced apoptosis by p33 ING1 required the presence of p53. Moreover, we found that p33 ING1 enhanced the expression of endogenous Bax and altered the mitochondrial membrane potential. Taken together, these observations strongly suggest that p33 ING1 cooperates with p53 in UVB-induced apoptosis via the mitochondrial cell death pathway in melanoma cells.

Human ING1 Proteins Differentially Regulate Histone Acetylation

ING1 proteins are nuclear, growth inhibitory, and regulate apoptosis in different experimental systems. Here we show that similar to their yeast homologs, human ING1 proteins interact with proteins associated with histone acetyltransferase (HAT) activity, such as TRRAP, PCAF, CBP, and p300. Human ING1 immunocomplexes contain HAT activity, and overexpression of p33(ING1b), but not of p47(ING1a), induces hyperacetylation of histones H3 and H4, in vitro and in vivo at the single cell level. p47(ING1a) inhibits histone acetylation in vitro and in vivo and binds the histone deacetylase HDAC1. Finally, we present evidence indicating that p33(ING1b) affects the degree of physical association between proliferating cell nuclear antigen (PCNA) and p300, an association that has been proposed to link DNA repair to chromatin remodeling. Together with the finding that human ING1 proteins bind PCNA in a DNA damage-dependent manner, these data suggest that ING1 proteins provide a direct linkage between DNA repair, apoptosis, and chromatin remodeling via multiple HAT.ING1.PCNA protein complexes.

The Tumor Suppressor ING1: Structure and Function

The biological functions of the tumor suppressor ING1 have been studied extensively in the past 5 years since it was cloned. Of the three alternatively spliced forms of ING1, p24ING1 has been the focus of much of past research. Information on the other currently known isoforms, p47ING1, p32ING1, and p27ING1, has been lacking. ING1 shares many biological functions with p53. It has been reported to mediate growth arrest, senescence, apoptosis, anchorage-dependent growth, and chemosensitivity. Some of these functions, such as cell-cycle arrest and apoptosis, have been shown to be dependent on the activity of both ING1 and p53 proteins. In this review, we will examine what is known about ING1 up to this point and clarify the cloning errors originating from the isolation of this gene.

UV induces nucleolar translocation of ING1 through two distinct nucleolar targeting sequences.

The ING1 candidate tumor suppressor is downregulated in a variety of primary tumors and established cancer cell lines. Blocking its expression experimentally promotes unregulated growth in vitro and in vivo, using cell and animal models. Alternative splicing products encode proteins that localize to the nucleus, inhibit cell cycle progression and affect apoptosis in different model systems. Here we show that ING1 proteins translocate to the nucleolus 12-48 h after UV-induced DNA damage. When a small 50 amino acid portion of ING1 was fused to green fluorescent protein, the fusion protein was efficiently targeted to the nucleolus, indicating that ING1 possesses an intrinsic nucleolar targeting sequence (NTS). We mapped this activity to two distinct 4 amino acid regions, which individually direct fused heterologous proteins to the nucleolus. Overexpression of ING1 induced apoptosis of primary fibroblasts in the presence and absence of UV exposure. In contrast, NTS mutants of ING1 that were not targeted to the nucleolus did not efficiently induce apoptosis when overexpressed and instead protected cells from UV-induced apoptosis. Taken together, these results indicate that UV induces ING1 to translocate to the nucleolus and that this translocation may facilitate apoptosis.

Expression of the novel tumour suppressor p33(ING1)is independent of p53.

A recently cloned tumour suppressor candidate, p33ING1, has been shown in vitro to collaborate with p53 to execute growth arrest and apoptosis. However, it is unclear as to how the expression of ING1 is regulated in normal and stress conditions. Using a p53-knockout mouse model, we investigated if the expression of ING1 was dependent on p53. We found that there was no difference in ING1 mRNA and protein levels between p53+/+ and p53–/– murine organs. In addition, when normal human epithelial keratinocytes (NHEK) and a keratinocyte cell line, HaCaT, which lacks wild-type p53 function, were exposed to UVB irradiation, the expression levels of ING1 were elevated in both NHEK and HaCaT cells. It is interesting, however, that UVB irradiation did not induce ING1 expression in dermal fibroblasts isolated from p53+/+ and p53–/– mice. Based on our findings, we therefore conclude that the expression of ING1 is independent of p53 status. UV induction of ING1 in keratinocytes suggests that ING1 may play a role in cellular stress response and skin carcinogenesis. © 2000 Cancer Research Campaign http://www.bjcancer.com

Genomic structure of the human ING1 gene and tumor-specific mutations detected in head and neck squamous cell carcinomas.

We characterized the genomic structure of the human ING1 gene, a candidate tumor suppressor gene, and found that the gene has three exons. We also demonstrated that four mRNA variants were transcribed from three different promoter regions. Of 34 informative cases of head and neck squamous cell carcinoma, 68% of tumors showed loss of heterozygosity at chromosome 13q33-34, where the ING1 gene is located. Here we present the first report that three missense mutations and three silent changes were detected in the ING1 gene in 6 of 23 tumors with allelic loss at the 13q33-34 region. These missense mutations were found within the PHD finger domain and nuclear localization motif in ING1 protein, probably abrogating the normal function.

A panel of CAb antibodies recognize endogenous and ectopically expressed ING1 protein.

Nine monoclonal antibodies (MAbs) against human and rodent ING1 protein have been generated using an IL6-secreting mouse myeloma line. These antibodies are all effective in recognizing ING1 protein in ELISAs, Western blot assays, and by indirect immunofluorescence. Combining different CAb monoclonal antibodies in a Western blot assay also allows detection of the very low levels of endogenous ING1 found in fibroblast cells in culture and the identification of at least two isoforms of ING1 in normal human diploid fibroblasts and established brain cancer cell lines.