A Novel Splice Variant of the Inhibitor of Growth 3 Lacks the Plant Homeodomain and Regulates Epithelial-Mesenchymal Transition in Prostate Cancer Cells.

Inhibitor of growth 3 (ING3) is one of five members of the ING tumour suppressor family, characterized by a highly conserved plant homeodomain (PHD) as a reader of the histone mark H3K4me3. ING3 was reported to act as a tumour suppressor in many different cancer types to regulate apoptosis. On the other hand, ING3 levels positively correlate with poor survival prognosis of prostate cancer (PCa) patients. In PCa cells, ING3 acts rather as an androgen receptor (AR) co-activator and harbours oncogenic properties in PCa. Here, we show the identification of a novel ING3 splice variant in both the human PCa cell line LNCaP and in human PCa patient specimen. The novel ING3 splice variant lacks exon 11, ING3∆ex11, which results in deletion of the PHD, providing a unique opportunity to analyse functionally the PHD of ING3 by a natural splice variant. Functionally, overexpression of ING3Δex11 induced morphological changes of LNCaP-derived 3D spheroids with generation of lumen and pore-like structures within spheroids. Since these structures are an indicator of epithelial-mesenchymal transition (EMT), key regulatory factors and markers for EMT were analysed. The data suggest that in contrast to ING3, ING3Δex11 specifically modulates the expression of key EMT-regulating upstream transcription factors and induces the expression of EMT markers, indicating that the PHD of ING3 inhibits EMT. In line with this, ING3 knockdown also induced the expression of EMT markers, confirming the impact of ING3 on EMT regulation. Further, ING3 knockdown induced cellular senescence via a pathway leading to cell cycle arrest, indicating an oncogenic role for ING3 in PCa. Thus, the data suggest that the ING3Δex11 splice variant lacking functional PHD exhibits oncogenic characteristics through triggering EMT in PCa cells.

Overexpression of ING3 is associated with attenuation of migration and invasion in breast cancer.

Inhibitor of growth 3 (ING3) has been identified as a potential cancer drug target, but little is known about its role in breast cancer. Thus, the present study aimed to investigate ING3 expression in breast cancer, its clinical value, and how ING3 influences the migration and invasion of breast cancer cells. The Cancer Genome Atlas and UALCAN databases were used to analyze ING3 expression in cancer tissues and normal tissues. Survival analysis was performed using the UALCAN, UCSC Xena and KM-plot databases. In addition, reverse transcription-quantitative PCR and western blot analyses were performed to detect ING3 mRNA and protein expression levels. ING3 was overexpressed via lentiviral vector transfection, while the Transwell and wound healing assays were performed to assess the cell migratory and invasive abilities. Protein interaction and pathway analyses were performed using the GeneMANIA and Kyoto Encyclopedia of Genes and Genomes databases, respectively. The results demonstrated that ING3 expression was significantly lower in cancer tissues compared with normal tissues (P<0.05). In addition, luminal A and human epidermal growth factor receptor 2 (HER2)-enriched breast cancer tissues expressed lower levels of ING3 compared with normal breast tissues. Notably, statistically significant differences were observed in long-term survival between patients with luminal A (P=0.04) and HER2-enriched (P=0.008) breast cancer, with high and low expression levels of ING3. The results of the Transwell migration and invasion assays demonstrated that overexpression of ING3 significantly inhibited the migratory and invasive abilities of MCF7 (P<0.05) and HCC1937 (P<0.05) cells. The results of the wound healing assay demonstrated that the percentage wound closure significantly decreased in cells transfected with LV5-ING3 compared with the negative control group at 12 h (P<0.05) and 24 h (P<0.01). The PI3K/AKT, JAK/STAT, NF-κB and Wnt/ß-catenin pathways are the potential pathways regulated by ING3. Notably, overexpression of ING3 inhibited migration and invasion in vitro. However, further studies are required to determine whether ING3 regulates the biological behavior of breast cancer via tumor-related pathways.

Nuclear ING3 Expression Is Correlated With a Good Prognosis of Breast Cancer.

The inhibitor of growth (ING) family was discovered as the type II tumor suppressors, which regulated the proliferation, apoptosis, differentiation, angiogenesis, metastasis, and invasion of tumor cells through multiple pathways. ING3, a new member of ING family, has been reported to be downregulated in several types of tumors. However, few studies on ING3 in breast cancer have been reported. In this study, we investigated the expression of ING3 and determined its prognostic value in breast cancer. The immunohistochemistry was performed to evaluate the expression of ING3 in tissue microarrays (TMA) including breast cancer tissues (n=211) and normal breast tissues (n=50). In normal breast tissues, ING3 protein was detected in both the cytoplasm and nucleus. In breast cancer tissues, ING3 protein was principally detected in the cytoplasm. Compared with normal breast tissues, the expression of ING3 in nucleus was remarkably reduced in breast cancer tissues. The downregulated ING3 in nucleus was significantly correlated with clinicopathological characteristics including histological grade, lymph node metastasis, and the status of ER and PR. In HER2 positive-type and triple-negative breast cancer (TNBC) patients, it had the lower rate of nuclear ING3 with high expression than that in luminal-type. Moreover, Kaplan-Meier curves demonstrated that the reduced expression of ING3 in nucleus was correlated with a poorer 5-DFS and 5-OS of breast cancer patients. Importantly, multivariate Cox regression analysis suggested that the reduced expression of ING3 in nucleus was an independent prognostic factor in breast cancer. Our study comprehensively described the expression of ING3 in breast cancer for the first time and proved that it was an independent prognostic predictor of breast cancer, as well as a new idea for study of breast cancer.

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.

Mechanism of Histone H3K4me3 Recognition by the Plant Homeodomain of Inhibitor of Growth 3.

Aberrant access to genetic information disrupts cellular homeostasis and can lead to cancer development. One molecular mechanism that regulates access to genetic information includes recognition of histone modifications, which is carried out by protein modules that interact with chromatin and serve as landing pads for enzymatic activities that regulate gene expression. The ING3 tumor suppressor protein contains a plant homeodomain (PHD) that reads the epigenetic code via recognition of histone H3 tri-methylated at lysine 4 (H3K4me3), and this domain is lost or mutated in various human cancers. However, the molecular mechanisms targeting ING3 to histones and the role of this interaction in the cell remain elusive. Thus, we employed biochemical and structural biology approaches to investigate the interaction of the ING3 PHD finger (ING3PHD) with the active transcription mark H3K4me3. Our results demonstrate that association of the ING3PHD with H3K4me3 is in the sub-micromolar range (KD ranging between 0.63 and 0.93 µm) and is about 200-fold stronger than with the unmodified histone H3. NMR and computational studies revealed an aromatic cage composed of Tyr-362, Ser-369, and Trp-385 that accommodate the tri-methylated side chain of H3K4. Mutational analysis confirmed the critical importance of Tyr-362 and Trp-385 in mediating the ING3PHD-H3K4me3 interaction. Finally, the biological relevance of ING3PHD-H3K4me3 binding was demonstrated by the failure of ING3PHD mutant proteins to enhance ING3-mediated DNA damage-dependent cell death. Together, our results reveal the molecular mechanism of H3K4me3 selection by the ING3PHD and suggest that this interaction is important for mediating ING3 tumor suppressive activities.