Gene Summary

Gene:ING4; inhibitor of growth family member 4
Aliases: my036, p29ING4
Summary:This gene encodes a tumor suppressor protein that contains a PHD-finger, which is a common motif in proteins involved in chromatin remodeling. This protein can bind TP53 and EP300/p300, a component of the histone acetyl transferase complex, suggesting its involvement in the TP53-dependent regulatory pathway. Multiple alternatively spliced transcript variants have been observed that encode distinct proteins. [provided by RefSeq, Jul 2008]
Databases:OMIM, HGNC, Ensembl, GeneCard, Gene
Protein:inhibitor of growth protein 4
Source:NCBIAccessed: 31 August, 2019


What does this gene/protein do?
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Cancer Overview

Research Indicators

Publications Per Year (1994-2019)
Graph generated 31 August 2019 using data from PubMed using criteria.

Literature Analysis

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Tag cloud generated 31 August, 2019 using data from PubMed, MeSH and CancerIndex

Specific Cancers (5)

Data table showing topics related to specific cancers and associated disorders. Scope includes mutations and abnormal protein expression.

Note: list is not exhaustive. Number of papers are based on searches of PubMed (click on topic title for arbitrary criteria used).

Latest Publications: ING4 (cancer-related)

Luo H, Chen Y, Niu L, et al.
Hepatoma Cell-Targeted Cationized Silk Fibroin as a Carrier for the Inhibitor of Growth 4-Interleukin-24 Double Gene Plasmid.
J Biomed Nanotechnol. 2019; 15(7):1622-1635 [PubMed] Related Publications
The construction of a targeted gene delivery system with low cytotoxicity to normal tissues is an urgent need for the clinical treatment of liver cancer. In this study,

Du Y, Cheng Y, Su G
The essential role of tumor suppressor gene
Biosci Rep. 2019; 39(1) [PubMed] Free Access to Full Article Related Publications
Inhibitor of growth 4 (ING4), a member of the ING family discovered in 2003, has been shown to act as a tumor suppressor and is frequently down-regulated in various human cancers. Numerous published

Bashanfer SAA, Saleem M, Heidenreich O, et al.
Disruption of MAPK1 expression in the ERK signalling pathway and the RUNX1‑RUNX1T1 fusion gene attenuate the differentiation and proliferation and induces the growth arrest in t(8;21) leukaemia cells.
Oncol Rep. 2019; 41(3):2027-2040 [PubMed] Related Publications
The t(8;21) translocation is one of the most frequent chromosome abnormalities associated with acute myeloid leukaemia (AML). This abberation deregulates numerous molecular pathways including the ERK signalling pathway among others. Therefore, the aim of the present study was to investigate the gene expression patterns following siRNA‑mediated suppression of RUNX1‑RUNX1T1 and MAPK1 in Kasumi‑1 and SKNO‑1 cells and to determine the differentially expressed genes in enriched biological pathways. BeadChip microarray and gene ontology analysis revealed that RUNX1‑RUNX1T1 and MAPK1 suppression reduced the proliferation rate of the t(8;21) cells with deregulated expression of several classical positive regulator genes that are otherwise known to enhance cell proliferation. RUNX1‑RUNX1T1 suppression exerted an anti‑apoptotic effect through the overexpression of BCL2, BIRC3 and CFLAR genes, while MAPK1 suppression induced apopotosis in t(8;21) cells by the apoptotic mitochondrial changes stimulated by the activity of upregulated TP53 and TNFSF10, and downregulated JUN gene. RUNX1‑RUNX1T1 suppression supported myeloid differentiation by the differential expression of CEBPA, CEBPE, ID2, JMJD6, IKZF1, CBFB, KIT and CDK6, while MAPK1 depletion inhibited the differentiation of t(8;21) cells by elevated expression of ADA and downregulation of JUN. RUNX1‑RUNX1T1 and MAPK1 depletion induced cell cycle arrest at the G0/G1 phase. Accumulation of cells in the G1 phase was largely the result of downregulated expression of TBRG4, CCNE2, FOXO4, CDK6, ING4, IL8, MAD2L1 and CCNG2 in the case of RUNX1‑RUNX1T1 depletion and increased expression of RASSF1, FBXO6, DADD45A and P53 in the case of MAPK1 depletion. Taken together, the current results demonstrate that MAPK1 promotes myeloid cell proliferation and differentiation simultaneously by cell cycle progression while suppresing apoptosis.

Yıldırım İH, Çakmak EA, Camcı C
Gene expression of ING4 gene in pancreatic cancers and splice forms of ING4 gene.
Cell Mol Biol (Noisy-le-grand). 2018; 64(13):6-9 [PubMed] Related Publications
Pancreatic cancer is characterized by rapid metastasis and resistant to medical treatments. As the other cancers, mutations of tumor suppressor genes that involved in suppression of cell growth are observed in pancreatic cancers. ING4 protein is one of the proteins involved in the regulation of p53 tumor suppressor gene functions. ING4 involved in suppression of cell proliferation, chromosome rearrangement, cell migration, and angiogenesis. In this study, gene expressions and splicing variants of ING4 gene were investigated. Fresh tumor and normal specimens of the same pancreatic cancer patients were used. Gene expression study carried out by calculating the brightness of the bands on agarose gel and splicing variants were detected by direct sequencing.  According to the results, three splice forms of ING4 and a decrease in gene expression of ING4 were determined. Splicing type of ING4 affects the translocation of ING4 proteins into the nucleus. To determine the gene expression of each splicing variant, will further clarify the role of ING4 in pancreatic cancers.

Gao J, Yang Y, Qiu R, et al.
Proteomic analysis of the OGT interactome: novel links to epithelial-mesenchymal transition and metastasis of cervical cancer.
Carcinogenesis. 2018; 39(10):1222-1234 [PubMed] Free Access to Full Article Related Publications
The role of O-GlcNAc transferase (OGT) in gene regulation and tumor invasion is poorly understood. Here, we have identified several previously undiscovered OGT-interacting proteins, including the PRMT5/WDR77 complex, the PRC2 complex, the ten-eleven translocation (TET) family, the CRL4B complex and the nucleosome remodeling and deacetylase (NuRD) complex. Genome-wide analysis of target genes responsive to OGT resulted in identification of a cohort of genes including SNAI1 and ING4 that are critically involved in cell epithelial-mesenchymal transition and invasion/metastasis. We have demonstrated that OGT promotes carcinogenesis and metastasis of cervical cancer cells. OGT's expression is significantly upregulated in cervical cancer, and low OGT level is correlated with improved prognosis. Our study has thus revealed a mechanistic link between OGT and tumor progression, providing potential prognostic indicators and targets for cancer therapy.

Jiang L, Zhang X, Xiang C, et al.
Differential cellular localization of CELSR2 and ING4 and correlations with hormone receptor status in breast cancer.
Histol Histopathol. 2018; 33(8):835-842 [PubMed] Related Publications
CELSR2 is postulated to be a receptor involved in contact-mediated communication; however, its expression and function in cancer remain unknown. ING4 is a tumor suppresor encoded by the ING4 gene which inhibits cell growth. The expression of CELSR2 and ING4 in breast tumors and in benign epithelial cells have been analyzed and correlated with HER2, ER, and PR status. Immunohistochemistry was used to analyze the expression of CELSR2 and ING4 protein in breast tumors and benign epithelial cells. The differential cellular localization of both markers was analyzed and results were also correlated with HER2, ER, and PR status. CELSR2 and ING4 cytoplasmic expression was significantly stronger in tumors than in benign epithelial cells, while the nuclear expression of both markers was significantly stronger in benign epithelial cells than in tumors. When comparing the two markers in the same type of tissues, the nuclear expression of CELSR2 was significantly stronger than cytoplasmic in benign epithelial cells, while there was no significant difference in the cellular localization of CELSR2 in tumors. For ING4, the cytoplasmic expression was significantly stronger than nuclear expression in tumors, while in benign epithelial cells, ING4 was expressed at similar levels in both compartments. There was no correlation between CELSR2 expression and HER2, ER, and PR status in tumors. However, the cytoplasmic expression of ING4 was associated with HER2 positivity in tumors. Both CELSR2 and ING4 display increased cytoplasmic staining in breast cancer cells compared to benign epithelium, suggesting a possible role of both genes in the pathogenesis of human mammary neoplasia.

Cai L, Li H, Chen C, et al.
Role of inhibitor of growth 4 in the suppression of human melanoma cells through the Fas/FasL-mediated apoptosis pathway.
Int J Mol Med. 2018; 41(2):1055-1061 [PubMed] Related Publications
Melanoma, the most aggressive form of skin cancer, is notoriously resistant to all current available therapies. Inhibitor of growth 4 (ING4), a novel member of the ING family of proteins, has previously been shown to play a critical role in the development of multiple tumors by regulating apoptosis, proliferation, cell cycle progress, migration and invasion. However, the functional role of ING4 in human melanoma remains unclear. To fully understand its potential role in human melanoma, in the present study, lentivirus (LV)‑ING4 and LV‑ING4‑short hairpin RNA were constructed and transfected into human melanoma A375 cells. First, the effect of overexpressing or downregulating ING4 on the apoptosis of the transfected melanoma cells and cluster of differentiation (CD)3+ T cells was investigated. In the present study, we found that the late apoptotic cells, and not the early apoptotic cells, were more in LV-ING4 group compared with LV-control, and both the early and late apoptosis of CD3+ T cells was significantly observed in A375 cells transfected with LV-ING4 compared with LV-control. Importantly, it was determined whether the overexpression of ING4 significantly induce apoptotic cell death via Fas/FasL (Fas death receptor/FasL) pathway activation and downregulation of poly(ADP‑ribose) polymerase, caspase‑3 and caspase‑8 in the melanoma cells and CD3+ T cells. These results demonstrated that overexpression of ING4 can induce the apoptosis of melanoma cells and CD3+ T cells through signaling pathways such as the Fas/FasL pathway, and that ING4 gene therapy for melanoma treatment is a novel approach.

Galal El-Shemi A, Mohammed Ashshi A, Oh E, et al.
Efficacy of combining ING4 and TRAIL genes in cancer-targeting gene virotherapy strategy: first evidence in preclinical hepatocellular carcinoma.
Gene Ther. 2018; 25(1):54-65 [PubMed] Free Access to Full Article Related Publications
Current treatments of hepatocellular carcinoma (HCC) are ineffective and unsatisfactory in many aspects. Cancer-targeting gene virotherapy using oncolytic adenoviruses (OAds) armed with anticancer genes has shown efficacy and safety in clinical trials. Nowadays, both inhibitor of growth 4 (ING4), as a multimodal tumor suppressor gene, and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), as a potent apoptosis-inducing gene, are experiencing a renaissance in cancer gene therapy. Herein we investigated the antitumor activity and safety of mono- and combined therapy with OAds armed with ING4 (Ad-ΔB/ING4) and TRAIL (Ad-ΔB/TRAIL) gene, respectively, on preclinical models of human HCC. OAd-mediated expression of ING4 or TRAIL transgene was confirmed. Ad-ΔB/TRAIL and/or Ad-ΔB/ING4 exhibited potent killing effect on human HCC cells (HuH7 and Hep3B) but not on normal liver cells. Most importantly, systemic therapy with Ad-ΔB/ING4 plus Ad-ΔB/TRAIL elicited more eradicative effect on an orthotopic mouse model of human HCC than their monotherapy, without causing obvious overlapping toxicity. Mechanistically, Ad-ΔB/ING4 and Ad-ΔB/TRAIL were remarkably cooperated to induce antitumor apoptosis and immune response, and to repress tumor angiogenesis. This is the first study showing that concomitant therapy with Ad-ΔB/ING4 and Ad-ΔB/TRAIL may provide a potential strategy for HCC therapy and merits further investigations to realize its possible clinical translation.

Sahin S, Ekinci O, Seckin S, Dursun A
The prognostic significance of ING4 expression on gastric gastrointestinal stromal tumors by immunohistochemistry.
Ann Ital Chir. 2017; 88:311-317 [PubMed] Related Publications
OBJECTIVE: Inhibitor of growth 4 (ING4) is a novel tumor suppressor gene that is reported to be down-regulated in various tumors including gastrointestinal stromal tumors (GISTs) originated from different locations, recently. Herein, we aimed to evaluate ING4 expression and its prognostic significance on gastric GISTs in order to add further data to the current literature.
MATERIAL AND METHODS: ING4 was evaluated in samples of gastric GISTs from 62 patients, by immunohistochemistry. The association between ING4 expression and clinicopathological features related with prognosis and overall survival (OS) were analyzed statistically.
RESULTS: There was statistically significant inverse correlation between ING4 expression and risk groups according to both NIH and AFIP, Ki67 index, tumor diameter, and mitotic count by univarite analysis (p=0.000, p=0.000, p=0.08, p=0.01, and p=0.028, respectively). The negative association between ING4 expression and risk groups according to both NIH (p=0.002, β=-0.263, t=-3.166) and AFIP (p=0.016, β=-0.244, t=-2.492) was supported by multivariate analysis. There was statistically significant direct correlation between low levels of ING4 expression and shorter OS by univariate (p=0.000) and multivariate analysis (p=0.000, β=0.769, t=9.798), as well as Kaplan-Meier method (p=0.035).
CONCLUSIONS: The low ING4 expression level was found to be related with unfavorable prognosis. Thus, we suggest that loss of ING4 expression might play a role in the progression of GISTs and might be used as a potential prognostic tool. Additionally, this is the first study that has evaluated the association of ING4 expression on gastric GISTs, to the best of our knowledge. Therefore, we claim that more comprehensive future studies including higher number of patients and longer follow-up might clarify the potential role of ING4 on pathogenesis and prognosis of GISTs.
KEY WORDS: Clinicopathological features, Gastrointestinal stromal tumor, ING4, Immunohistochemistry.

Wang S, Reinhard S, Li C, et al.
Antitumoral Cascade-Targeting Ligand for IL-6 Receptor-Mediated Gene Delivery to Glioma.
Mol Ther. 2017; 25(7):1556-1566 [PubMed] Free Access to Full Article Related Publications
The effective treatment of glioma is largely hindered by the poor transfer of drug delivery systems across the blood-brain barrier (BBB) and the difficulty in distinguishing healthy and tumorous cells. In this work, for the first time, an interleukin-6 receptor binding I

Zhai Q, Wang L, Zhao P, Li T
Role of citrullination modification catalyzed by peptidylarginine deiminase 4 in gene transcriptional regulation.
Acta Biochim Biophys Sin (Shanghai). 2017; 49(7):567-572 [PubMed] Related Publications
Peptidylarginine deiminase 4 (PADI4), a new histone modification enzyme, which converts both arginine and monomethyl-arginine to citrulline, has gained massive attention in recent years as a potential regulator of gene transcription. Recent studies have shown that arginine residues R2, R8, R17, and R26 in the H3 tail and R3 in the H4 tail can be deiminated by PADI4. This kind of histone post-translational modification has the potential to antagonize histone methylation and coordinate with histone deacetylation to regulate gene transcription. PADI4 also deiminates non-histone proteins, such as p300, NPM1, ING4, RPS2, and DNMT3A. PADI4 has been shown to involve in cell apoptosis and differentiation. Moreover, PADI4 can interact with tumor suppressor p53 and regulate the transcriptional activity of p53. Dysregulation of PADI4 is implicated in a variety of diseases, including rheumatoid arthritis, tumor development, and multiple sclerosis. A wide variety of PADI4 inhibitors have been identified. Further understanding of PADI4 functions may lead to novel diagnostic and therapeutic approaches in these diseases. This review summarizes the recent progress in the study of the regulation mechanism of PADI4 on gene transcription and the major physiological functions of PADI4 in human diseases.

Lango-Chavarría M, Chimal-Ramírez GK, Ruiz-Tachiquín ME, et al.
A 22q11.2 amplification in the region encoding microRNA-650 correlates with the epithelial to mesenchymal transition in breast cancer primary cultures of Mexican patients.
Int J Oncol. 2017; 50(2):432-440 [PubMed] Free Access to Full Article Related Publications
Breast cancer ranks first in incidence and mortality in working age women. Cancer initiation and progression relies on accumulation of genetic and epigenetic aberrations that alter cellular processes, among them, epithelial to mesenchymal transition (EMT) denotes particularly aggressive neoplasias given its capacity to invade and metastasize. Several microRNAs (miRNA) have been found able to regulate gene expression at the core of EMT. In this study, the Affymetrix CytoScan HD array was used to analyze three different primary tumor cell isolates from Mexican breast cancer patients. We found an amplification in band 22q11.2 shared by the three samples, in the region that encodes miRNA-650. Overexpression of this miRNA has been associated with downregulation of tumor suppressors ING4 and NDRG2, which have been implicated in cancer progression. Using the Pathway Linker platform the ING4 and NDRG2 interaction networks showed a significant association with signaling pathways commonly deregulated in cancer. Also, several studies support their participation in the EMT. Supporting the latter, we found that the three primary isolates were E-cadherin negative, vimentin positive, presented a cancer stem cell-like phenotype CD44+CD24-/low and were invasive in Transwell invasion assays. This evidence suggests that the gain of region 22q11.2 contributes to trigger EMT. This is the first evidence linking miR-650 and breast cancer.

Hu X, Feng Y, Sun L, et al.
Roles of microRNA-330 and Its Target Gene ING4 in the Development of Aggressive Phenotype in Hepatocellular Carcinoma Cells.
Dig Dis Sci. 2017; 62(3):715-722 [PubMed] Related Publications
BACKGROUND: Aberrant expression of microRNAs contributes to tumor growth and progression.
AIMS: This study was designed to explore the prognostic and biological significance of miR-330 in hepatocellular carcinoma (HCC).
METHODS: The expression of miR-330 and its associations with tumor parameters and overall survival were analyzed in HCC patients. The biological functions of miR-330 in HCC cell growth, invasion, and tumorigenesis were investigated. Bioinformatic analysis and luciferase reporter assays were performed to search for potential targets of miR-330.
RESULTS: The miR-330 level was significantly higher in HCCs than in adjacent normal tissues (P = 0.0085). High expression of miR-330 was significantly associated with more aggressive phenotypes and shorter overall survival in HCC. Loss- and gain-of-function studies indicated the favorable effect of miR-330 on tumor cell growth, invasion, and tumorigenesis. Inhibitor of growth 4 (ING4) was identified to be a direct target of miR-330. Overexpression of miR-330 reduced the expression of ING4 in HCC cells. Importantly, restoration of ING4 almost completely reversed the promotion of HCC cell proliferation and invasion by miR-330.
CONCLUSIONS: Altogether, this study demonstrates that upregulation of miR-330 is associated with poor prognosis and contributes to more aggressive phenotypes of HCC. The oncogenic role of miR-330 in HCC is linked to downregulation of ING4.

Chen Y, Huang Y, Hou P, et al.
ING4 suppresses tumor angiogenesis and functions as a prognostic marker in human colorectal cancer.
Oncotarget. 2016; 7(48):79017-79031 [PubMed] Free Access to Full Article Related Publications
ING4, a potential tumor suppressor, is implicated in cell cycle arrest, apoptosis, cell migration and angiogenesis. Here, we investigated the clinical value of ING4 and its impact on angiogenesis in colorectal cancer (CRC). In this study, we found that ING4 expression was significantly reduced in CRC tissues versus paired normal colon tissues. Moreover, low ING4 expression was significantly associated with increased lymph node metastasis, advanced TNM stage and poor overall survival. Multivariate Cox regression analysis showed that ING4 expression was an independent favourable prognostic factor for CRC (hazard ratio = 0.45, P = 0.001). In addition, we found that ING4 strongly inhibited CRC angiogenesis by suppressing Sp1 expression and transcriptional activity through ubiquitin degradation and down-regulating the expressions of Sp1 downstream pro-angiogenic genes, MMP-2 and COX-2. Moreover, ING4 might inhibit phosphorylation activity of cyclin/CDK2 complexes to trigger Sp1 degradation by inducing p21 expression in despite of p53 status. Our findings imply that reduced ING4 expression in CRC resulted in increased angiogenesis and contributed to CRC metastasis and poor prognosis. Restoration of ING4 may be a novel strategy for the treatment of metastatic CRC.

Li ZY, Li QZ, Chen L, et al.
Histone Deacetylase Inhibitor RGFP109 Overcomes Temozolomide Resistance by Blocking NF-κB-Dependent Transcription in Glioblastoma Cell Lines.
Neurochem Res. 2016; 41(12):3192-3205 [PubMed] Related Publications
Glioblastoma (GBM) is the most frequent and aggressive tumour in the central nervous system. Many studies have demonstrated that upregulation of the NF-κB onco-pathway is accompanied by the acquisition of Temozolomide (TMZ) resistance in GBM cells. Here, we show that RGFP109, a selective histone deacetylase (HDAC1 and HDAC3) inhibitor, overcomes TMZ resistance and downregulates the expression of NF-κB-regulated pro-survival genes in a TMZ-resistant (TR) GBM cell line. RGFP109 did not alter the phosphorylation levels of NF-κB/p65 or inhibitory κBα (IκBα). Immunofluorescence microscopy showed that RGFP109 does not block the nuclear translocation of NF-κB/p65. However, co-immunoprecipitation assays revealed that RGFP109 induces the hyperacetylation of NF-κB/p65 and histones, and blocks interactions between NF-κB/p65 and its coactivators, p300 and p300/CBP-associated factor (PCAF). These results indicate that RGFP109-mediated post-translational nuclear acetylation may be involved in the regulation of NF-κB. Electrophoretic mobility shift assays revealed that RGFP109 reduces NF-κB/p65 binding to κB-DNA and decreased the transcriptional level of κB-mediated genes, suggesting that RGFP109-induced hyperacetylation leads to attenuated transcription of the κB gene. In addition, RGFP109 elevates the expression of inhibitor of growth 4 (ING4), which is typically downregulated in GBM cells. Importantly, we found that RGFP109 enhances ING4 recognition and binding to NF-κB/p65, which may be positively correlated with reduced interactions between NF-κB/p65 and p300/PCAF, thereby effecting transcription of the κB gene. Finally, we show that knockdown of ING4 with plasmids containing pcDNA3.1-ING4 shRNA abolished the effect of RGFP109. Therefore, ING4 may act as a corepressor and facilitate RGFP109-triggered suppression of the NF-κB pathway. Taken together, our data show that RGFP109, an HDAC inhibitor, in combination with TMZ may be a therapeutic candidate for patients with temozolomide-resistant GBM.

Zhang R, Jin J, Shi J, Hou Y
INGs are potential drug targets for cancer.
J Cancer Res Clin Oncol. 2017; 143(2):189-197 [PubMed] Related Publications
PURPOSE: The inhibitor of growth (ING) family consists of ING1, ING2, ING3, ING4 and ING5, which function as the type II tumor suppressors. INGs regulate cell proliferation, senescence, apoptosis, differentiation, angiogenesis, DNA repair, metastasis, and invasion by multiple pathways. In addition, INGs increase cancer cell sensitivity for chemotherapy and radiotherapy, while clinical observations show that INGs are frequently lost in some types of cancers. The aim of the study was to summarize the recent progress regarding INGs regulating tumor progression.
METHODS: The literatures of INGs regulating tumor progression were searched and assayed.
RESULTS: The regulating signaling pathways of ING1, ING2, ING3 or ING4 on tumor progression were shown. The mechanisms of INGs on tumor suppression were also assayed.
CONCLUSIONS: This review better summarized the signaling mechanism of INGs on tumor suppression, which provides a candidate therapy strategy for cancers.

Ren X, Liu H, Zhang M, et al.
Co-expression of ING4 and P53 enhances hypopharyngeal cancer chemosensitivity to cisplatin in vivo.
Mol Med Rep. 2016; 14(3):2431-8 [PubMed] Free Access to Full Article Related Publications
Hypopharyngeal cancer is a distinct type of malignant head and neck tumor, which exhibits low sensitivity to anti-cancer drugs. The importance of developing methods for reducing chemotherapy resistance, and improving and enhancing prognosis has previously been emphasized and is considered a challenge for effective clinical treatment of hypopharyngeal cancer. The current study investigated the effects of co‑expression of inhibitor of growth protein 4 (ING4) and P53, a tumor suppressor gene, on chemosensitivity to cisplatin in human hypopharyngeal cancer xenografts in vivo, and the potential molecular mechanisms involved. A tumor model was established by injecting athymic nude mice with FADU human hypopharyngeal cancer cells. Five days after intratumoral and peritumoral injections of an empty adenoviral vector (Ad), Ad‑ING4‑P53, cisplatin, or a combination of Ad‑ING4‑P53 and cisplatin (Ad‑ING4‑P53 + cisplatin) every other day for 5 days, the mice were euthanized and their tumors, livers, and kidneys were removed. The tumor weights were used to calculate the inhibition rate, and the expression levels of ING4 and P53 were detected by reverse transcription‑polymerase chain reaction. Additionally, apoptotic cells were detected using terminal deoxynucleotidyl transferase dUTP nick end labeling, and immunohistochemistry determined the levels ING4, P53, B‑cell lymphoma‑2 (Bcl‑2) and Bcl‑2 associated X protein (Bax) protein expression. The results demonstrated increased expression of ING4 and P53 in the Ad‑ING4‑P53 groups compared with PBS and Ad groups, indicating successful introduction of the genes into the tumor cells. Notably, the Ad‑ING4‑P53 + cisplatin group exhibited a higher inhibition rate compared with the four other groups. The results of immunohistochemistry analysis demonstrated that Bax expression was increased and Bcl‑2 was decreased in the Ad‑ING4‑P53 + cisplatin group. This suggested that the enhanced cisplatin chemosensitivity with Ad-ING4-P53 gene therapy in hypopharyngeal cancer xenografts may be associated with apoptosis induction through upregulation of Bax expression and downregulation of Bcl‑2. The results of the present study indicated that gene therapy combined with cisplatin treatment may be a promising treatment for human hypopharyngeal cancer.

Li S, Zeng A, Hu Q, et al.
miR-423-5p contributes to a malignant phenotype and temozolomide chemoresistance in glioblastomas.
Neuro Oncol. 2017; 19(1):55-65 [PubMed] Free Access to Full Article Related Publications
BACKGROUND: Gliomas are based on a genetic abnormality and present with a dismal prognosis. MicroRNAs (miRNAs) are considered to be important mediators of gene expression in glioma tissues.
METHODS: Real-time PCR was used to analyze the expression of microRNA-423-5p (miR-423-5p) in human glioma samples and normal brain tissue. Apoptosis, cell cycle, proliferation, immunostaining, transwell, in vitro 2D and 3D migration, and chemosensitivity assays were performed to assess the phenotypic changes in glioma cells overexpressing miRNA-423-5p. Western blotting was used to determine the expression of inhibitor of growth 4 (ING-4)in glioma tissues, and a luciferase reporter assay was conducted to confirm whether ING-4 is a direct target of miR-423-5p. Western blotting was used to identify the potential signaling pathways that are affected in glioma cell growth by miR-423-5p. Xenograft tumors were examined in vivo for the carcinogenic effects of miR-423-5p in glioma tissues.
RESULTS: We first reported that miR-423-5p expression was increased in gliomas and was a potential tumor promoter via targeting ING-4. The overexpression of miR-423-5p resulted in upregulation of important signaling molecules such as p-AKT and p-ERK1/2. In clinical samples, miR-423-5p was dysregulated, and a corresponding alteration in ING-4 expression was observed (P = .0207). Furthermore, the overexpression of miR-423-5p strengthened glioma cell proliferation, angiogenesis, and invasion. Finally, miR-423-5p overexpression also strengthened GBM neurosphere formation and rendered glioma cells resistant to temozolomide (TMZ).
CONCLUSION: This study establishes that miR-423-5p functions as an oncogene in glioma tissues by suppressing ING-4 and suggests that it has therapeutic potential for glioma.

Rakshit N, Yang S, Zhou W, et al.
Adenovirus-mediated co-expression of ING4 and PTEN cooperatively enhances their antitumor activity in human hepatocellular carcinoma cells.
Acta Biochim Biophys Sin (Shanghai). 2016; 48(8):704-13 [PubMed] Related Publications
Both inhibitor of growth 4 (ING4) and phosphatase and tensin homolog deleted on chromosome 10 (PTEN) are well known as tumor suppressors that are closely related to tumor occurrence and progression. It was reported that ING4 and PTEN showed synergistic antitumor activities in nasopharyngeal carcinoma cells. The two tumor suppressors demonstrated synergistic effect on growth inhibition and apoptosis activation. In this study, we investigated their therapeutic potential in hepatocellular carcinoma (HCC) cells. Recombinant adenoviruses co-expressing ING4 and PTEN (Ad-ING4-PTEN) were constructed, and the antitumor effect on SMMC-7721 and HepG2 HCC cells was evaluated. Ad-ING4-PTEN cooperatively inhibited cell growth, stimulated apoptosis, and suppressed invasion in both HCC cells, and regulated cell cycle in SMMC-7721. Further studies showed that the combination of ING4 and PTEN by Ad-ING4-PTEN cooperatively enhanced the alteration of the expression of cell cycle-related proteins (p53, p21, and cyclin D1) and apoptotic factors (Bad, Bcl-2, Bcl-XL, and Bax), which are involved in the regulation of cell cycle and the activation of apoptotic pathways, leading to the synergistic antitumor effect. These results indicate that the combination of ING4 and PTEN may provide an effective therapeutic strategy for HCC.

Pan X, Wang R, Bian H, et al.
Overexpression of Inhibitor of Growth 4 Enhances Radiosensitivity in Non-Small Cell Lung Cancer Cell Line SPC-A1.
Technol Cancer Res Treat. 2017; 16(5):533-545 [PubMed] Free Access to Full Article Related Publications
Inhibitor of growth 4 is a member of the inhibitor of growth family proteins, which is involved in cell apoptosis, migration, invasion, and cell cycle progress. In this study, we investigated the inhibitor of growth 4 level in non-small cell lung cancer tissues and explored the antitumor activity of inhibitor of growth 4 in vitro and in vivo using non-small cell lung cancer cell line SPC-A1 and its underlying molecular mechanisms. We also explored its role on the radiosensitivity in SPC-A1 cells. The level of inhibitor of growth 4 protein was significantly decreased in 28 cases of non-small cell lung cancer tissues in comparison with corresponding noncancerous lung epithelial tissues. Upregulation of inhibitor of growth 4 by plasmid pcDNA3.1-ING4 delivery could suppress proliferation and increase apoptosis of SPC-A1 cells both in vitro and in vivo. Additionally, we found that overexpression of inhibitor of growth 4 in SPC-A1 cell line could lead to a higher Bcl-2/Bax ratio, which might be an important factor in the apoptosis regulation. Furthermore, overexpression of inhibitor of growth 4 enhanced the radiosensitivity of SPC-A1 cells to irradiation. Inhibitor of growth 4 upregulation plus radiotherapy induced synergistic tumor suppression in SPC-A1 xenografts implanted in athymic nude mice. Thus, the restoration of inhibitor of growth 4 function might provide a potential strategy for non-small cell lung cancer radiosensitization.

Ashshi AM, El-Shemi AG, Dmitriev IP, et al.
Combinatorial strategies based on CRAd-IL24 and CRAd-ING4 virotherapy with anti-angiogenesis treatment for ovarian cancer.
J Ovarian Res. 2016; 9(1):38 [PubMed] Free Access to Full Article Related Publications
BACKGROUND: A major hurdle incurrent to the human clinical application of conditionally replicative adenovirus (CRAd)-based virotherapy agents is their limited therapeutic efficacy. In this study we evaluated whether arming our previously reported Ad5/3Δ24 CRAd vector containing a 24-base pair deletion in the E1A conserved region 2, which allows selective replication within Rb-p16-deficient tumor cells, to express therapeutic genes could improve oncolytic virus potency in ovarian cancer cells. We choose to assess the therapeutic benefits achieved by virus-mediated expression of interleukin 24 (IL-24), a cytokine-like protein of the IL-10 family, and the inhibitor of growth 4 (ING4) tumor suppressor protein.
RESULTS: The generated CRAd-IL24 and CRAd-ING4 vectors were tested in ovarian cancer cell lines in vitro to compare their replication, yield, and cytotoxic effects with control CRAd Ad5/3∆24 lacking the therapeutic gene. These studies showed that CRAd-IL24 infection resulted in significantly increased yield of infectious particles, which translated to a marked enhancement of virus-induced cytotoxic effects as compared to CRAd-ING4 and non-armed CRAd. Testing CRAd-IL24 and CRAd-ING4 vectors combined together did not revealed synergistic effects exceeding oncolytic potency of single CRAD-IL24 vector. Both CRAds were also tested along with anti-VEGF monoclonal antibody Avastin and showed no significant augmentation of viral cytolysis by anti-angiogenesis treatment in vitro.
CONCLUSIONS: Our studies validated that arming with these key immunomodulatory genes was not deleterious to virus-mediated oncolysis. These findings thus, warrant further preclinical studies of CRAd-IL24 tumoricidal efficacy in murine ovarian cancer models to establish its potential utility for the virotherapy of primary and advanced neoplastic diseases.

Meng Y, Wang S, Li C, et al.
Photothermal combined gene therapy achieved by polyethyleneimine-grafted oxidized mesoporous carbon nanospheres.
Biomaterials. 2016; 100:134-42 [PubMed] Related Publications
Combining controllable photothermal therapy and efficacious gene therapy in a single platform holds great promise in cancer therapy due to the enhanced combined therapeutic effects. Herein, polyethyleneimine-grafted oxidized mesoporous carbon nanospheres (OP) were developed for combined photothermal combined gene therapy in vitro and in vivo. The synthesized OP was characterized to have three dimensional spherical structure with uniformed diameter, ordered mesopores with graphitic domains, high water dispersion with zeta potential of +22 mV, and good biocompatibility. Consequently, OP was exploited as the photothermal convertor with strong NIR absorption and the gene vector via electrostatic interaction, which therefore cannot only deliver the therapeutic gene (pING4) to tumors for gene therapy, but also can eliminate the tumors by photothermal ablation. Moreover, the improved gene therapy accompanied by the NIR photothermally enhanced gene release was also well achieved based on OP. The excellent combined therapeutic effects demonstrated in vitro and in vivo suggested the OP's potential for cancer therapy.

Ma Y, Cheng X, Wang F, et al.
ING4 Inhibits Proliferation and Induces Apoptosis in Human Melanoma A375 Cells via the Fas/Caspase-8 Apoptosis Pathway.
Dermatology. 2016; 232(3):265-72 [PubMed] Related Publications
BACKGROUND: Inhibitor of growth 4 (ING4) plays a role in regulating the cell cycle, apoptosis, cell invasion and migration, but the mechanisms involved remain to be elucidated.
OBJECTIVE: To explore how ING4 affects human malignant melanoma A375 cells.
METHODS: Recombinant lentiviral vectors (A375/pLenO-GTP-ING4) were constructed and transfected into A375 cells (experimental group). The impact of ING4 on the proliferation and apoptosis of A375 cells was investigated in in vitro and in vivo experiments in mice using the MTT assay and flow cytometry.
RESULTS: In the experimental group, optical density was lower and apoptotic cells were more frequent from days 2-5 (p = 0.000 and p < 0.01); there were smaller xenografts and more apoptotic cells in mice (all p < 0.05); moreover, increased levels of Fas, cleaved caspase-8 and caspase-3, and decreased levels of FasL and procyclic acidic repetitive protein were observed in vitro and in vivo.
CONCLUSION: ING4 might suppress proliferation and enhance apoptosis in human malignant melanoma cells by activating Fas-induced apoptosis in a caspase-8-dependent pathway.

Qu H, Yin H, Yan S, et al.
Inhibitor of growth 4 suppresses colorectal cancer growth and invasion by inducing G1 arrest, inhibiting tumor angiogenesis and reversing epithelial-mesenchymal transition.
Oncol Rep. 2016; 35(5):2927-35 [PubMed] Related Publications
Previous studies have found that inhibitor of growth 4 (ING4), a tumor suppressor, is reduced in human colorectal cancer (CRC), and is inversely correlated with clinical Dukes' stage, histological grade, lymph node metastasis and microvessel density (MVD). However, its underlying mechanism remains undetermined. In the present study, we analyzed ING4 expression in a panel of human CRC cells using low (LS174T and SW480) and high (LoVo and SW620) metastatic cell lines. We demonstrated that both the low and high metastatic CRC cells exhibited a lower level of ING4 compared to the level in normal human colorectal mucous epithelial FHC cells. Furthermore, ING4 expression in high metastatic CRC cells was less than that in low metastatic CRC cells. We then generated a lentivirus construct expressing ING4 and green fluorescent protein (GFP), established a ING4-stably transgenic LoVo CRC cell line, and investigated the effect of lentiviral-mediated ING4 expression on high metastatic LoVo CRC cells. Gain-of-function studies revealed that ING4 significantly inhibited LoVo CRC cell growth and invasion in vitro and induced cell cycle G1 phase arrest. Moreover, ING4 obviously suppressed LoVo CRC subcutaneously xenografted tumor growth and reduced tumor MVD in vivo in athymic BALB/c nude mice. Mechanistically, ING4 markedly upregulated P21 and E-cadherin but downregulated cyclin E, interleukin (IL)-6, IL-8, vascular endothelial growth factor (VEGF), Snail1, N-cadherin and vimentin in the LoVo CRC cells. Our data provide compelling evidence that i) ING4 suppresses CRC growth possibly via induction of G1 phase arrest through upregulation of P21 cyclin-dependent kinase (CDK) inhibitor and downregulation of cyclin E as well as inhibition of tumor angiogenesis through reduction of IL-6, IL-8 and VEGF proangiogenic factors; ii) ING4 inhibits CRC invasion and metastasis probably via a switch from mesenchymal marker N-cadherin to epithelial marker E-cadherin through downregulation of Snail1 epithelial-mesenchymal transition (EMT)-inducing transcription factor (EMT-TF).

Kuninty PR, Bojmar L, Tjomsland V, et al.
MicroRNA-199a and -214 as potential therapeutic targets in pancreatic stellate cells in pancreatic tumor.
Oncotarget. 2016; 7(13):16396-408 [PubMed] Free Access to Full Article Related Publications
Pancreatic stellate cells (PSCs) are the key precursor cells for cancer-associated fibroblasts (CAFs) in pancreatic tumor stroma. In this study, we explored miRNA as therapeutic targets in tumor stroma and found miR-199a-3p and miR-214-3p induced in patient-derived pancreatic CAFs and TGF-β-activated human PSCs (hPSCs). Inhibition of miR-199a/-214 using hairpin inhibitors significantly inhibited TGFβ-induced differentiation markers (e.g. α-SMA, collagen, PDGFβR), migration and proliferation. Furthermore, heterospheroids of Panc-1 and hPSCs attained smaller size with hPSCs transfected with anti-miR-199a/-214 compared to control anti-miR. The conditioned medium obtained from TGFβ-activated hPSCs induced tumor cell growth and endothelial cell tube formation. Interestingly, these inductions were abrogated in hPSCs transfected with anti-miR-199a or miR-214. Moreover, IPA analyses revealed signaling pathways related to miR-199a (TP53, mTOR, Smad1) and miR-214 (PTEN, Bax, ING4). Taken together, this study reveals miR-199a-3p and miR-214-3p as major regulators of PSC activation and PSC-induced pro-tumoral effects, representing them as key therapeutic targets in pancreatic cancer.

Yuan S, Jin J, Shi J, Hou Y
Inhibitor of growth-4 is a potential target for cancer therapy.
Tumour Biol. 2016; 37(4):4275-9 [PubMed] Related Publications
The inhibitor of growth-4 (ING-4) belongs to the inhibitor of growth (ING) family that is a type II tumor suppressor gene including five members (ING1-5). As a tumor suppressor, ING4 inhibits tumor growth, invasion, and metastasis by multiple signaling pathways. In addition to that, ING4 can facilitate cancer cell sensitivity to chemotherapy and radiotherapy. Although ING4 loss is observed for many types of cancers, increasing evidences show that ING4 can be used for gene therapy. In this review, the recent progress of ING4 regulating tumorigenesis is discussed.

Zhang H, Zhou X, Xu C, et al.
Synergistic tumor suppression by adenovirus-mediated ING4/PTEN double gene therapy for gastric cancer.
Cancer Gene Ther. 2016; 23(1):13-23 [PubMed] Related Publications
Both inhibitor of growth 4 (ING4) and phosphatase and tensin homolog (PTEN) have been shown to be strong candidate tumor suppressors. However, the combined efficacy of ING4 and PTEN for human gastric cancer remains to be determined. In this report, we constructed a multiple promoter expression cassette-based recombinant adenovirus coexpressing ING4 and PTEN (AdVING4/PTEN), assessed the combined effects of AdVING4/PTEN on gastric cancer using wild-type p53 AGS and SNU-1 human gastric cancer cell lines, and elucidated its underlying mechanisms. We found that AdVING4/PTEN-induced synergistic growth inhibition and apoptosis in vitro AGS or SNU-1 tumor cells and in vivo AGS xenografted tumors subcutaneously inoculated in athymic BALB/c nude mice. Mechanistically, AdVING4/PTEN exhibited an enhanced effect on upregulation of p53, Ac-p53 (K382), P21, Bax, PUMA, Noxa, cleaved Caspase-9, cleaved Caspase-3 and cleaved PARP as well as downregulation of Bcl-2 in vitro and in vivo. In addition, AdVING4/PTEN synergistically downregulated tumor vessel CD34 expression and reduced microvessel density, and additively inhibited vascular endothelial growth factor (VEGF) expression in vivo. The synergistic tumor suppression elicited by AdVING4/PTEN was closely associated with the synergistic induction of apoptosis possibly via enhancement of endogenous p53 responses through cooperatively facilitating p53's stability and acetylation, and the synergistic inhibition of tumor angiogenesis probably via overlapping reduction of VEGF through cooperatively downregulating hypoxia inducible factor-1α's level and transcription activity. Thus, our results indicate that cancer gene therapy combining ING4 and PTEN may constitute a novel and effective therapeutic modality for human gastric cancer and other cancers.

Sun J, Shen Q, Lu H, et al.
Oncogenic Ras suppresses ING4-TDG-Fas axis to promote apoptosis resistance.
Oncotarget. 2015; 6(39):41997-2007 [PubMed] Free Access to Full Article Related Publications
Ras is aberrantly activated in many cancers and active DNA demethylation plays a fundamental role to establish DNA methylation pattern which is of importance to cancer development. However, it was unknown whether and how Ras regulate DNA demethylation during carcinogenesis. Here we found that Ras downregulated thymine-DNA glycosylase (TDG), a DNA demethylation enzyme, by inhibiting the interaction of transcription activator ING4 with TDG promoter. TDG recruited histone lysine demethylase JMJD3 to the Fas promoter and activated its expression, thus restoring sensitivity to apoptosis. TDG suppressed in vivo tumorigenicity of xenograft pancreatic cancer. Thus, we speculate that reversing Ras-mediated ING4 inhibition to activate Fas expression is a potential therapeutic approach for Ras-driven cancers.

Wu J, Zhu Y, Xu C, et al.
Adenovirus-mediated p53 and ING4 gene co-transfer elicits synergistic antitumor effects through enhancement of p53 acetylation in breast cancer.
Oncol Rep. 2016; 35(1):243-52 [PubMed] Related Publications
Multigene-based combination therapy may be an effective practice in cancer gene therapy. Substantial studies have demonstrated that tumor suppressor p53 acetylation is indispensable for p53 activation. Inhibitor of growth 4 (ING4), as a novel tumor suppressor, is capable of remarkably enhancing p53 acetylation and its transcriptional activity. Hence, we assumed that combined treatment of p53 and ING4 double tumor suppressors would exhibit enhanced antitumor effects. The combined therapeutic efficacy of p53 and ING4 for human cancers has not been previously reported. We thus generated multiple promoter expression cassette-based recombinant adenovirus-co-expressing ING4 and p53 double tumor suppressor genes (AdVING4/p53), evaluated the combined effects of AdVING4/p53 on breast cancer using the MDA-MB-231 (mutant p53) human breast cancer cell line, and also elucidated its underlying molecular mechanisms. We demonstrated that AdVING4/p53-mediated p53 and ING4 co-expression induced synergistic growth inhibition and apoptosis as well as enhanced effects on upregulation of acetylated p53, P21, Bax, PUMA, Noxa, cleaved caspase-9, cleaved caspase-3 and cleaved PARP, and downregulation of Bcl-2, CD31 and microvessel density (MVD) in MDA-MB-231 breast cancer in vitro and/or in vivo subcutaneous (s.c.) xenografted tumors. The synergistic antitumor activity elicited by AdVING4/p53 was closely associated with the enhanced activation of the intrinsic apoptotic pathway and synergistic inhibition of tumor angiogenesis, very possibly via ING4-mediated enhancement of p53 acetylation and activity. Thus, our results indicate that cancer gene therapy combining two or more tumor suppressors such as p53 and ING4 may constitute a novel and effective therapeutic modality for human breast cancer and other cancers.

Yan A, Yang C, Chen Z, et al.
MiR-761 Promotes Progression and Metastasis of Non-Small Cell Lung Cancer by Targeting ING4 and TIMP2.
Cell Physiol Biochem. 2015; 37(1):55-66 [PubMed] Related Publications
BACKGROUND/AIMS: The aim of this study was to investigate the role of microRNA miR-761 in the progression and metastasis of non-small cell lung cancer (NSCLC), and the mechanisms by which miR-761 regulates cell proliferation and metastatic activity of NSCLC cell lines.
METHODS: Quantitative real-time PCR (qRT-PCR) was used to assess miR-761 expression in NSCLC serum and tissue. MTT, wound healing, and transwell assays were performed to examine the role of miR-761 in regulation of cell proliferation and metastatic activity in NSCLC cell lines. In addition, the correlations of miR-761 expression with clinical-pathologic factors were statistically analyzed. Finally, we investigated whether miR-761 promotes proliferation and metastasis in NSCLC cell lines by targeting ING4 (inhibitor of growth family, member 4) and TIMP2 (tissue inhibitor of metalloproteinase 2).
RESULTS: MiR-761 was significantly upregulated in both NSCLC serum and tissues as compared to normal participants and paired noncancerous tissues respectively. Ectopic expression of miR-761 promoted cell proliferation and metastasis in H460 cells, while miR-761 inhibitor reduced proliferation rates and metastasis in H23 cells. Furthermore, luciferase reporter assay and functional analyses indicated that miR-761 directly targeted ING4 and TIMP2.
CONCLUSION: miR-761 promotes progression and metastasis of NSCLC by targeting ING4 and TIMP2.

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