Expanding the molecular spectrum of gene fusions in endometrial stromal sarcoma: Novel subunits of the chromatin remodeling complexes PRC2 and NuA4/TIP60 as alternative fusion partners.

Endometrial stromal sarcomas (ESS) are morphologically and molecularly heterogeneous. We report novel gene fusions (EPC1::EED, EPC1::EZH2, ING3::PHF1) identified by targeted RNA sequencing in five cases. The ING3::PHF1-fusion positive ESS presented in a 58-year-old female as extrauterine mesocolonic, ovarian masses, and displayed large, monomorphic ovoid-to-epithelioid cells arranged in solid sheets. The patient remained alive with disease 13 months after surgery. The three ESS with EPC1::EED occurred in the uterine corpus in patients with a median age of 58 years (range 27-62 years). One tumor showed a uniform epithelioid nested morphology, while the other two were composed of monomorphic spindle cells in fascicles with elevated mitotic figures, focal tumor cell necrosis, and lymphovascular invasion. At a median follow-up of 20 months, two patients developed local recurrence, including one with concomitant distant metastasis, while one patient remained free of disease. All three patients were alive at the last follow-up. The EPC1::EZH2-fusion positive ESS presented in a 52-year-old female in the uterus, and displayed uniform spindled cells arranged in short fascicles, with focally elevated mitotic activity but without necrosis. The patient remained free of disease 3 months after surgery. All cases were diffusely positive for CD10; four diffusely express estrogen and progesterone receptors. Our study expands the molecular spectrum of EPC1 and PHF1-related gene fusions in ESS to include additional novel subunits of the PRC2 and/or NuA4/TIP60 complexes. These cases displayed a monomorphic epithelioid or spindled phenotype, spanning low-grade and high-grade cytomorphology, all expressing CD10 and commonly ER and PR, and are prone to local and/or distant spread.

Nuclear localization of ING3 is required to suppress melanoma cell migration, invasion and angiogenesis.

Inhibitor of growth family member 3 (ING3), a tumor suppressor, plays crucial roles in cell cycle regulation, apoptosis and transcription. Previous studies suggest important roles of nuclear ING3, however, the nuclear localization sequence (NLS) of ING3 is not defined and its biological functions remain to be elucidated. In this study, various ING3 mutants were generated to identify its NLS. The NLS of ING3 was determined as KKFK between 164 and 167 amino acids. More intriguingly, replacement of Lysine 164 residue of ING3 with alanine (K164A) resulted in retention of ING3 in the cytoplasm. Overexpression of ING3 led to inhibition of melanoma cell migration, invasion, and angiogenesis respectively, however, this inhibition was abrogated in cells with overexpression of ING3-K164A mutant. In conclusion, this study identified the NLS of ING3 and demonstrated the significance of ING3 nuclear localization for tumor suppressive functions of ING3, and future studies await to elucidate the role of ING3 (K164) post-modificaton in its nuclear transportation and cancer development.

Overexpression of miR-101-2 in donor cells improves the early development of Holstein cow somatic cell nuclear transfer embryos.

Accumulating studies have suggested that microRNA play a part in regulating multiple cellular processes, such as cell proliferation, apoptosis, the cell cycle, and embryo development. This study explored the effects of miR-101-2 on donor cell physiological status and the development of Holstein cow somatic cell nuclear transfer (SCNT) embryos in vitro. Holstein cow bovine fetal fibroblasts (BFF) overexpressing miR-101-2 were used as donor cells to perform SCNT; then, cleavage rate, blastocyst rate, inner cell mass-to-trophectoderm ratio, and the expression of some development- and apoptosis-related genes in different groups were analyzed. The miR-101-2 suppressed the expression of inhibitor of growth protein 3 (ING3) at mRNA and protein levels, expedited cell proliferation, and decreased apoptosis in BFF, suggesting that ING3, a target gene of miR-101-2, is a potential player in this process. Moreover, by utilizing donor cells overexpressing miR-101-2, the development of bovine SCNT embryos in vitro was significantly enhanced; the apoptotic rate in SCNT blastocysts was reduced, and the inner cell mass-to-trophectoderm ratio and SOX2, POU5F1, and BCL2L1 expression significantly increased, whereas BAX and ING3 expression decreased. Collectively, these findings suggest that miR-101-2 promotes BFF proliferation and vitality, reduces their apoptosis, and improves the early development of SCNT embryos.

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.

ING3 is associated with increased cell invasion and lethal outcome in ERG-negative prostate cancer patients.

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.

Tumor suppressor gene ING3 induces cardiomyocyte hypertrophy via inhibition of AMPK and activation of p38 MAPK signaling.

Cardiac hypertrophy, an adaptive growth process that occurs in response to various pathophysiological stimuli, constitutes an important risk factor for the development of heart failure. However, the molecular mechanisms that regulate this cardiac growth response are not completely understood. Here we revealed that ING3 (inhibitor of growth family, member 3), a type II tumor suppressor, plays a critical role in the regulation of cardiac hypertrophy. ING3 expression was present in relatively high abundance in the heart, and was prominently upregulated in hypertrophic agonists angiotensin II (Ang II), phenylephrine (PE), or isoproterenol (ISO)-stimulated cardiomyocytes and in hearts of rat undergoing abdominal aortic constriction (AAC) surgery. In cardiomyocytes, overexpression of ING3 caused an increase in ANP, BNP and ß-MHC mRNA levels and cell surface area, while depletion of ING3 attenuated PE-induced cardiomyocyte hypertrophy. Mechanistically, we have demonstrated that overexpression of ING3 could inactivate the AMPK and activate the canonical p38 MAPK signaling. Remarkably, AMPK agonist AICAR or p38 MAPK inhibitor SB203580 abrogated ING3-induced hypertrophic response in cardiomyocytes. In summary, our data disclose a novel role of ING3 as an inducer of pathological cardiac hypertrophy, suggesting that silencing of ING3 may be explored as a potential therapeutic target in preventing cardiac hypertrophy.

ING3 inhibits the malignant progression of lung adenocarcinoma by negatively regulating ITGB4 expression to inactivate Src/FAK signaling.

Lung adenocarcinoma (LUAD) is the most commonly diagnosed subtype of lung cancer worldwide. Inhibitor of growth 3 (ING3) serves as a tumor suppressor in many cancers. This study aimed to elucidate the role of ING3 in the progression of LUAD and investigate the underlying mechanism related to integrin ß4 (ITGB4) and Src/focal adhesion kinase (FAK) signaling. ING3 expression in LUAD tissues and the correlation between ING3 expression and prognosis were analyzed by bioinformatics databases. After evaluating ING3 expression in LUAD cells, ING3 was overexpressed to assess the proliferation, cell cycle arrest, migration and invasion of LUAD cells. Then, ITGB4 was upregulated to observe the changes of malignant activities in ING3-overexpressed LUAD cells. The transplantation tumor model of NCI-H1975 cells in nude mice was established to analyze the antineoplastic effect of ING3 upregulation in vivo. Downregulated ING3 expression was observed in LUAD tissues and cells and lower ING3 expression predicated the poor prognosis. ING3 upregulation restrained the proliferation, migration, invasion and induced the cell cycle arrest of NCI-H1975 cells. Additionally, ITGB4 expression was negatively correlated with ING3 expression in LUAD tissue. ING3 led to reduced expression of ITGB4, Src and p-FAK. Moreover, ITGB4 overexpression alleviated the effects of ING3 upregulation on the malignant biological properties of LUAD cells. It could be also found that ING3 upregulation limited the tumor volume, decreased the expression of ITGB4, Src and p-FAK, which was restored by ITGB4 overexpression. Collectively, ING3 inhibited the malignant progression of LUAD by negatively regulating ITGB4 expression to inactivate Src/FAK signaling.

Structural analysis of ING3 protein and histone H3 binding.

Proteins belonging to the ING family regulate the transcriptional state of chromatin by recruiting remodeling complexes to sites with histone H3 trimethylated at Lysine 4 (H3K4me3). This modification is recognized by the Plant HomeoDomain (PHD) present at the C-terminal region of the five ING proteins. ING3 facilitates acetylation of histones H2A and H4 by the NuA4-Tip60 MYST histone acetyl transferase complex, and it has been proposed to be an oncoprotein. The crystal structure of the N-terminal domain of ING3 shows that it forms homodimers with an antiparallel coiled-coil fold. The crystal structure of the PHD is similar to those of its four homologs. These structures explain the possible deleterious effects of ING3 mutations detected in tumors. The PHD binds histone H3K4me3 with low-micromolar, and binds the non-methylated histone with a 54-fold reduced affinity. Our structure explains the impact of site directed mutagenesis experiments on histone recognition. These structural features could not be confirmed for the full-length protein as solubility was insufficient for structural studies, but the structure of its folded domains suggest a conserved structural organization for the ING proteins as homodimers and bivalent readers of the histone H3K4me3 mark.

Inhibitor of Growth Factors Regulate Cellular Senescence.

The Inhibitor of Growth (ING) proteins are a group of tumor suppressors with five conserved genes. A common motif of ING factors is the conserved plant homeodomain (PHD), with which they bind to chromatin as readers of the histone mark trimethylated histone H3 (H3K4me3). These genes often produce several protein products through alternative splicing events. Interestingly, ING1 and ING2 participate in the establishment of the repressive mSIN3a-HDAC complexes, whereas ING3, ING4, and ING5 are associated with the activating HAT protein complexes. In addition to the modulation of chromatin's structure, they regulate cell cycle transition, cellular senescence, repair of DNA damage, apoptosis, and angiogenic pathways. They also have fundamental effects on regulating cellular senescence in cancer cells. In the current review, we explain their role in cellular senescence based on the evidence obtained from cell line and animal studies, particularly in the context of cancer.

Loss of Ing3 Expression Results in Growth Retardation and Embryonic Death.

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.

Focus-ING on DNA Integrity: Implication of ING Proteins in Cell Cycle Regulation and DNA Repair Modulation.

The ING family of tumor suppressor genes is composed of five members (ING1-5) involved in cell cycle regulation, DNA damage response, apoptosis and senescence. All ING proteins belong to various HAT or HDAC complexes and participate in chromatin remodeling that is essential for genomic stability and signaling pathways. The gatekeeper functions of the INGs are well described by their role in the negative regulation of the cell cycle, notably by modulating the stability of p53 or the p300 HAT activity. However, the caretaker functions are described only for ING1, ING2 and ING3. This is due to their involvement in DNA repair such as ING1 that participates not only in NERs after UV-induced damage, but also in DSB repair in which ING2 and ING3 are required for accumulation of ATM, 53BP1 and BRCA1 near the lesion and for the subsequent repair. This review summarizes evidence of the critical roles of ING proteins in cell cycle regulation and DNA repair to maintain genomic stability.

Exploiting ING2 Epigenetic Modulation as a Therapeutic Opportunity for Non-Small Cell Lung Cancer.

Non-small cell lung cancer (NSCLC) has been the leading cause of cancer-related death worldwide, over the last few decades. Survival remains extremely poor in the metastatic setting and, consequently, innovative therapeutic strategies are urgently needed. Inhibitor of Growth Gene 2 (ING2) is a core component of the mSin3A/Histone deacetylases complex (HDAC), which controls the chromatin acetylation status and modulates gene transcription. This gene has been characterized as a tumor suppressor gene and its status in cancer has been scarcely explored. In this review, we focused on ING2 and other mSin3A/HDAC member statuses in NSCLC. Taking advantage of existing public databases and known pharmacological properties of HDAC inhibitors, finally, we proposed a therapeutic model based on an ING2 biomarker-guided strategy.

Long non-coding RNA CASC7 inhibits the proliferation and migration of colon cancer cells via inhibiting microRNA-21.

Recent evidences highlight the crucial regulatory roles of long noncoding RNAs (lncRNA) in tumor biology. LncRNA CASC7 is a ∼9.3kb lncRNA whose function is currently unknown. The present study aimed to investigate the expression of CASC7 in patients with colorectal cancer (CRC) and its effect on CRC cells. The expression levels of CASC7, miR-21 and ING3 were estimated by reverse transcription-quantitative polymerase chain reaction (RT-PCR) or western blot in CRC tissues and CRC cell lines (SW480 and HCT-116). The relationship between miR-21 and CASC7 or ING3 was analyzed by RNA immunoprecipitation (RIP), RNA pull-down and luciferase reporter assay. In addition, the biological roles of CASC7 were examined using cell counting kit-8 assay, flow cytometry, and migration and invasion assays following the downregulation or upregulation of CASC7 by small interfering RNA or pcDNA-CASC7, respectively. In this study, CASC7 expression was significantly decreased in CRC tissues and CRC cell lines. Further functional experiments suggested that CASC7 overexpression could inhibit cell viability, migration and invasion, and promote apoptosis in CRC cells. CASC7 and ING3 were both a target of miR-21 in CRC cells, and CASC7 could control ING3 expression by regulating miR-21. Moreover, we have found that CASC7 inhibited colon cancer cell proliferation and migration via miR-21/ING3 axis. These observations suggested that CASC7 played an important role in CRC pathogenesis and may be considered as a novel diagnostic marker of CRC.

Biochemical and molecular evidences for the antitumor potential of Ginkgo biloba leaves extract in rodents.

Hepatocellular carcinoma (HCC) is one of the deadliest primary cancers, with a 5-year survival rate of 10% or less. This study was undertaken to elucidate the underlying biochemical and molecular mechanisms in favor of N-nitrosodiethylamine-induced hepatocellular carcinoma. Furthermore, the aim of this work was extended to explore the efficacy of Ginkgo biloba leaves extract in deterioration of HCC in rats. In the current study, HCC group experienced significant downregulation of ING-3 gene expression and upregulation of Foxp-1 gene expression in liver. Treatment of HCC groups with Ginkgo biloba leaves extract resulted in upregulation of ING-3 and downregulation of Foxp-1 gene expression in liver. In addition, there was significant increase in serum alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA) and glypican-3 (GPC-3) levels in HCC group versus the negative control group. In contrast, the groups with HCC subjected to either high or low dose of Ginkgo biloba leaves extract elicited significant reduction (P<0.05) of AFP, CEA and GPC-3 in serum compared to the untreated HCC rats. Besides, histological examination of liver tissue sections of rats in HCC group revealed typical anaplasia. Interestingly, treatment with Ginkgo biloba leaves extract elicited marked improvement in the histological feature of liver tissue in HCC groups. In conclusion, this research indicated that the carcinogenic potency of N-nitrosodiethylamine targeted multiple systems on the cellular and molecular levels. In addition, the results of the current study shed light on the promising anticancer activity of Ginkgo biloba leaves extract in treatment of hepatocellular carcinoma induced chemically in the experimental model through its apoptotic and antiproliferative properties.

The expression and signi ficance of ING3 in colorectal cancer / 实用肿瘤学杂志

Objetcvi e Inhibitor of growth TNG3 is a candidate tumor suppressor gene ,which belongs to the human ING gene family .It has been interested by scientist because of its putative roles as tumor suppressors . There is a low expression level of ING3 in a variety of tumors.However,the relationship of ING3 and the develop-ment of colorectal cancer is unclear yet .The aim of this studyis to examine the expression of ING 3 in colorectal cancer by qRT-PCR.Methods Statistical analysis was performed with the expression of ING 3 mRNA which has been evaluated in 82 matched samples comprising primary colorectal cancer and paired non -cancerous mu-cosa samples.Results In 51%colorectal cancer patients ,the expression level of ING3 was significantly lower in cancer tissue than that in normal tissue .The expression level of ING3 was closely related to pT、pM stage , and TNM stage(P<0.05).The Univariate analysis showed that the pT、pN、pM stage,TNMstage and ING3 expression levels were associated with disease -free survival(P<0.05).CRC patients with low -expression level of ING3 showed shorter disease -free survivals(P<0.05).Conclusion The expression of ING3 might contribute to the CRC development or progression .