ING3 promotes prostate cancer growth by activating the androgen receptor

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

Ubiquitin-specific protease 12 interacting partners Uaf-1 and WDR20 are potential therapeutic targets in prostate cancer

The androgen receptor (AR) is a key transcription factor in the initiation and progression of prostate cancer (PC) and is a major therapeutic target for the treatment of advanced disease. Unfortunately, current therapies are not curative for castration resistant PC and a better understanding of AR regulation could identify novel therapeutic targets and biomarkers to aid treatment of this disease. The AR is known to be regulated by a number of post-translational modifications and we have recently identified the deubiquitinating enzyme Usp12 as a positive regulator of AR. We determined that Usp12 deubiquitinates the AR resulting in elevated receptor stability and activity. Furthermore, Usp12 silencing was shown to reduce proliferation of PC cells. Usp12 is known to require the co-factors Uaf-1 and WDR20 for catalytic activity. In this report we focus further on the role of Uaf-1 and WDR20 in Usp12 regulation and investigate if these co-factors are also required for controlling AR activity. Firstly, we confirm the presence of the Usp12/Uaf-1/WDR20 complex in PC cells and demonstrate the importance of Uaf-1 and WDR20 for Usp12 stabilisation. Consequently, we show that individual silencing of either Uaf-1 or WDR20 is sufficient to abrogate the activity of the Usp12 complex and down-regulate AR-mediated transcription via receptor destabilisation resulting in increased apoptosis and decreased colony forming ability of PC cells. Moreover, expression of both Uaf-1 and WDR20 is higher in PC tissue compared to benign controls. Overall these results highlight the potential importance of the Usp12/Uaf-1/WDR20 complex in AR regulation and PC progression. Androgen receptor is a key transcriptional regulator in prostate cancer. Usp12/Uaf-1/WDR20 complex plays a crucial role in androgen receptor stability and activity. Destabilising an individual Usp12/Uaf-1/WDR20 complex member reduces the protein levels of the whole complex and diminishes androgen receptor activity. Protein levels of all members of the Usp12/Uaf-1/WDR20 complex are significantly increased in PC

Human ex vivo prostate tissue model system identifies ING3 as an oncoprotein

Background: Although the founding members of the INhibitor of Growth (ING) family of histone mark readers, ING1 and ING2, were defined as tumour suppressors in animal models, the role of other ING proteins in cellular proliferation and cancer progression is unclear. Methods: We transduced ex vivo benign prostate hyperplasia tissues with inducible lentiviral particles to express ING proteins. Proliferation was assessed by H3S10phos immunohistochemistry (IHC). The expression of ING3 was assessed by IHC on a human prostate cancer tissue microarray (TMA). Gene expression was measured by DNA microarray and validated by real-time qPCR. Results: We found that ING3 stimulates cellular proliferation in ex vivo tissues, suggesting that ING3 could be oncogenic. Indeed, ING3 overexpression transformed normal human dermal fibroblasts. We observed elevated levels of ING3 in prostate cancer samples, which correlated with poorer patient survival. Consistent with an oncogenic role, gene-silencing experiments revealed that ING3 is required for the proliferation of breast, ovarian, and prostate cancer cells. Finally, ING3 controls the expression of an intricate network of cell cycle genes by associating with chromatin modifiers and the H3K4me3 mark at transcriptional start sites. Conclusions: Our investigations create a shift in the prevailing view that ING proteins are tumour suppressors and redefine ING3 as an oncoprotein

Molecular mechanism of the TP53-MDM2-AR-AKT signalling network regulation by USP12

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

The Role of the ING3 Epigenetic Regulator in Prostate Cancer

INhibitor of growth (ING) proteins are epigenetic regulators and stoichiometric members of histone acetyltransferase (KAT) or histone deacetylase (KDAC) complexes. By reading the histone mark H3K4me3, they direct their complexes to chromatin to alter gene expression. This thesis focuses on the role of ING3 in prostate cancer biology. Since rigorous characterization of antibodies is a prerequisite to acquire reliable results, we began by characterizing a new mouse monoclonal antibody against ING3. We profiled the expression of ING3 protein in normal human tissues and found that it is highly expressed in bone marrow, suggesting high expression in hematopoietic cell precursors. We also reported that ING3 protein levels are highest in proliferating tissues of the small intestine and epidermis. These data suggest a role for ING3 in promoting cell growth and renewal. In the second part of this study, we investigated the effects of ING3 on the androgen receptor (AR) pathway in prostate cancer (PC). We hypothesized that ING3 by virtue of being an essential member of TIP60 KAT complex, plays a role in post-translational modifications of AR protein and thereby contributes to PC progression. We found that the levels of ING3 and AR are positively correlated in patient samples and cell lines. ING3 potentiates androgen effects, activating expression of androgen responsive genes and AR-regulated reporters. We showed that ING3 interacts with the binding domain of AR and this interaction happens in the cytoplasm in the absence of androgens. ING3 increases AR-TIP60 interaction, promoting AR acetylation and nuclear translocation. The activating role of ING3 is independent of its ability to target the TIP60 complex to H3K4me3, identifying a previously unknown function for ING3. Knockdown of ING3 inhibits PC cell proliferation and migration, establishing ING3 as a positive regulator of growth in PC. Lastly, we asked whether ING3 could serve as a biomarker to distinguish latent versus aggressive PC. ING3 levels are higher in aggressive PC, with high levels of ING3 predicting shorter overall survival. Analysis with other predictive factors shows that including ING3 levels provides more accurate prognosis in PC

Biological Functions of the ING Proteins

The proteins belonging to the inhibitor of growth (ING) family of proteins serve as epigenetic readers of the H3K4Me3 histone mark of active gene transcription and target histone acetyltransferase (HAT) or histone deacetylase (HDAC) protein complexes, in order to alter local chromatin structure. These multidomain adaptor proteins interact with numerous other proteins to facilitate their localization and the regulation of numerous biochemical pathways that impinge upon biological functions. Knockout of some of the ING genes in murine models by various groups has verified their status as tumor suppressors, with ING1 knockout resulting in the formation of large clear-cell B-lymphomas and ING2 knockout increasing the frequency of ameloblastomas, among other phenotypic effects. ING4 knockout strongly affects innate immunity and angiogenesis, and INGs1, ING2, and ING4 have been reported to affect apoptosis in different cellular models. Although ING3 and ING5 knockouts have yet to be published, preliminary reports indicate that ING3 knockout results in embryonic lethality and that ING5 knockout may have postpartum effects on stem cell maintenance. In this review, we compile the known information on the domains of the INGs and the effects of altering ING protein expression, to better understand the functions of this adaptor protein family and its possible uses for targeted cancer therapy

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

Epigenetically decipherING the genome: A role for PHDs

International audienceWhile completion of the initial phase of the human genome project redefined genetics over two decades ago, the field of epigenetics is currently redefining how chromatin structure affects the gene expression to mold the proteome and the cell phenotype. Posttranslational modification (PTM) of nucleic acids and proteins is central to chromatin organization and helps govern transcription and translation to affect specific phenotypes. Zinc fingers are a common constituent of chromatin regulators and the plant homeodomain (PHD) forms of zinc finger function as readers of the histone code. Understanding the structural mechanisms driving the recognition of PTMs by PHDs is at the core of efforts to understand how chromatin architecture is maintained and modulated. Here, we describe the roles of PHD finger proteins in chromatin remodeling through their specific binding to PTMs on histone proteins with an emphasis on the Inhibitor of Growth (ING) family. Once bound to methylated chromatin, ING1-5 family members serve as scaffolds for the docking of complexes of effector proteins that are vital to gene-specific activation and/or inactivation events that occur within chromatin. All members of the ING family recognize different methylated forms of lysine 4 near the amino terminus of core histone 3 (H3K4me1/2/3), but different members target either histone acetyltransferase (HAT) or histone deacetylase (HDAC) complexes to regulate histone acetylation and subsequently chromatin structure and gene expression. ING-mediated changes in gene expression have now been shown to profoundly affect a broad range of biological processes including development, differentiation, cellular senescence, and oncogenesis. © 2024 Elsevier Inc. All rights reserved