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

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

Ex vivo Culture and Lentiviral Transduction of Benign Prostatic Hyperplasia (BPH) Samples.

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

Differential effects of transforming growth factor-beta 1 on the expression of matrix metalloproteinases and tissue inhibitors of metalloproteinases in young and old human fibroblasts

The balance between the activities of matrix metalloproteinases (MMPs) and the tissue inhibitors of metalloproteinases (TIMPs) is an important control point in tissue remodeling. Previous studies have demonstrated elevated expression of the MMPs collagenase and stromelysin-1 by aged human diploid fibroblasts compared to early-passage cultures. We show here that aging cells display an altered response to transforming growth factor-beta 1 (TGF beta 1) that selectively affects MMP mRNA expression. In both young and old cells, phorbol myristoyl-13 acetate (PMA) induced the expression of transcripts of collagenase, stromelysin-1, gelatinase-B, TIMP-1, and TIMP-3. In young cells, TGF beta 1 reciprocally modulated PMA-induced MMP and TIMP gene expression leading to reduced levels of transcripts for the MMPs and augmented accumulation of TIMP-1 and TIMP-3 mRNAs. However, repressing effects of TGF beta 1 on collagenase, stromelysin-1, and gelatinase-B RNA expression were not apparent in old cells, though induction of the TIMP genes was unimpaired. By electrophoretic mobility shift analysis the nuclear transcription factors AP1 and serum response factor (SRF) showed reduced levels of DNA binding activities in old fibroblasts compared to young cells. A probe for the TGF beta-inhibitory element (TIE) gave equivalent levels of complexes with nuclear extracts from both types of cells, though of different mobilities. We conclude that the effects of TGF beta 1 on MMP and TIMP gene expression involve different cellular intermediaries, and suggest that altered composition or modification of TIE binding factors in aging cells may underlie the failure of TGF beta 1-mediated transcription repression. This mechanism may contribute to elevated constitutive expression of MMPs in old cells and to the connective tissue deterioration that accompanies the aging process