ING4 Expression Research Articles

Decreased Nuclear Immunoexpression of ING3 is a Frequent Event in Lip Carcinogenesis.

Evaluate the immunohistochemical expression of the ING3 in actinic cheilitis and squamous cell carcinoma of the lower lip. Forty-five specimens of actinic cheilitis and 48 specimens of squamous cell carcinoma of the lower lip were submitted to immunohistochemical detection of ING3. The protein expression in different cellular sublocations was compared between the two groups, and associations with the clinicopathological variables were analyzed. A significance level of 5% was adopted for all tests. Deaths were significantly more frequent in tumors with a high histopathological risk score (p < 0.05). In actinic cheilitis, significant differences were found in the nucleus-cytoplasmic expression of ING3 and expression restricted to the cytoplasm with binary histopathological grading (p < 0.05). In squamous cell carcinoma of the lower lip, there was no statistically significant difference when comparing ING3 expressions with clinical and morphological parameters (p > 0.05). Nucleo-cytoplasmic ING3 expression was significantly lower in squamous cell carcinoma of the lower lip when compared to actinic cheilitis (p < 0.05) and the expression restricted to the cytoplasm was significantly higher in squamous cell carcinoma of the lower lip (p < 0.05). The results of this study suggest that there is a marked decrease in the nuclear expression of ING3 as malignant progression occurs, indicating an impaired tumor suppressor function of this protein in actinic cheilitis and squamous cell carcinoma of the lower lip.

MicroRNA-650 promotes melanoma metastasis via targeting inhibitor of growth family member 4

ObjectiveThis study aimed to evaluate the effects of microRNA-650 (miR-650) on melanoma metastasis and reveal the regulatory relationship between miR-650 and the inhibitor of growth family member 4 (ING4). MethodsmiR-650 expression was determined in human melanoma WM115 and A-375 cells. WM115 cells were transfected with miR-650 mimic or mimic control. The invasion and migration abilities of transfected WM115 cells were analyzed using Transwell and wound healing assays, respectively. Then, miR-650-overexpression lentivirus vector was constructed and transfected into WM115 cells. After injection into the mice, the number of micro-metastatic foci in the lung tissues was counted. A regulatory relationship between miR-650 and ING4 was identified in WM115 and A-375 cells. ResultsThe miR-650 expression was upregulated in WM115 and A-375 cells. WM115 cells transfected with the miR-650 mimic exhibited higher invasive and migratory abilities than mock cells or cells transfected with negative control (NC). The number of micro-metastatic foci was significantly higher in mice injected with Lenti-miR-650 than that in those injected with mock or NC controls. Transfection with miR-650 mimic observably inhibited the expression of ING4 in WM115 and A-375 cells, whereas transfection with miR-650 inhibitor had the opposite effect. Dual-luciferase reporter gene assay showed that the miR-650 mimic inhibited the luciferase activity of ING4. ConclusionmiR-650 promotes melanoma metastasis by downregulating ING4 expression.

ING1 inhibits Twist1 expression to block EMT and is antagonized by the HDAC inhibitor vorinostat

ING1 is a chromatin targeting subunit of the Sin3a histone deacetylase (HDAC) complex that alters chromatin structure to subsequently regulate gene expression. We find that ING1 knockdown increases expression of Twist1, Zeb 1&2, Snai1, Bmi1 and TSHZ1 drivers of EMT, promoting EMT and cell motility. ING1 expression had the opposite effect, promoting epithelial cell morphology and inhibiting basal and TGF-β-induced motility in 3D organoid cultures. ING1 binds the Twist1 promoter and Twist1 was largely responsible for the ability of ING1 to reduce cell migration. Consistent with ING1 inhibiting Twist1 expression in vivo, an inverse relationship between ING1 and Twist1 levels was seen in breast cancer samples from The Cancer Genome Atlas (TCGA). The HDAC inhibitor vorinostat is approved for treatment of multiple myeloma and cutaneous T cell lymphoma and is in clinical trials for solid tumours as adjuvant therapy. One molecular target of vorinostat is INhibitor of Growth 2 (ING2), that together with ING1 serve as targeting subunits of the Sin3a HDAC complex. Treatment with sublethal (LD25-LD50) levels of vorinostat promoted breast cancer cell migration several-fold, which increased further upon ING1 knockout. These observations indicate that correct targeting of the Sin3a HDAC complex, and HDAC activity in general decreases luminal and basal breast cancer cell motility, suggesting that use of HDAC inhibitors as adjuvant therapies in breast cancers that are prone to metastasize may not be optimal and requires further investigation.

Expression and prognostic value of ING3 in advanced laryngeal squamous cell carcinoma

Objectives: Laryngeal squamous cell carcinomas (LSCC) is one of the most common aggressive neoplasms of the head and neck region. There is a significant need for identification of successful and accurate prognostic markers to better estimate the clinical outcomes for LSCC patients. In this study, we aimed at analyzing the differential expressions of inhibitor growth (ING) family members and to evaluate the prognostic values of deregulated ING genes in LSCC. Methods: We investigated the relative expressions of ING genes in laryngeal tumor-normal tissue pairs in the mRNA level using quantitative real-time polymerase chain reaction and relative expression of ING3 in the protein level using Western Blot analysis. Results: The rate of genetic alterations of ING3 was relatively higher in head and neck cancer including LSCC. ING3 expression was significantly upregulated in LSCC tissue samples in both mRNA and protein level. Higher expression of ING3 was also correlated with poor disease-free survival of patients with head and neck cancer. Conclusions: Our findings assigned an oncogenic feature for ING3 in laryngeal cancer with a significant upregulation detected in advanced cases and suggested a vital prognostic potential for ING3.

The roles of ING5 in cancer: A tumor suppressor.

As a Class II tumor suppressor, ING5 contains nuclear localization signal, plant homeodomain, novel conserved region, and leucine zipper-like domains. ING5 proteins form homodimer into a coil-coil structure, and heterodimers with ING4, histone H3K4me3, histone acetyltransferase (HAT) complex, Tip60, Cyclin A1/CDK2, INCA1 and EBNA3C for the transcription of target genes. The acetylated proteins up-regulated by ING5 are preferentially located in nucleus and act as transcription cofactors, chromatin and DNA binding functions, while those down-regulated by ING5 mostly in cytoplasm and contribute to metabolism. ING5 promotes the autoacetylation of HAT p300, p53, histone H3 and H4 for the transcription of downstream genes (Bax, GADD45, p21, p27 and so forth). Transcriptionally, YY1 and SRF up-regulate ING5 mRNA expression by the interaction of YY1-SRF-p53-ING5 complex with ING5 promoter. Translationally, ING5 is targeted by miR-196, miR-196a, miR-196b-5p, miR-193a-3p, miR-27-3p, miR-200b/200a/429, miR-1307, miR-193, miR-222, miR-331-3p, miR-181b, miR-543 and miR-196-b. ING5 suppresses proliferation, migration, invasion and tumor growth of various cancer cells via the suppression of EGFR/PI3K/Akt, IL-6/STAT3, Akt/NF-κB/NF-κB/MMP-9 or IL-6/CXCL12 pathway. ING5-mediated chemoresistance is closely linked to anti-apoptosis, overexpression of chemoresistant genes, the activation of PI3K/Akt/NF-κB and Wnt/β-catenin signal pathways. Histologically, ING5 abrogation in gastric stem-like and pdx1-positive cells causes gastric dysplasia and cancer, and conditional ING5 knockout in pdx1-positive and gastric chief cells increases MNU-induced gastric carcinogenesis. Intestinal ING5 deletion increases AOM/DSS- induced colorectal carcinogenesis and decreases high-fat-diet weight. The overexpression and nucleocytoplasmic translocation of ING5 are seen during carcinogenesis, and ING5 expression was inversely associated with aggressive behaviors and poor prognosis in a variety of cancers. These findings indicated that ING5 might be used for a molecular marker for carcinogenesis and following progression, and as a target for gene therapy if its chemoresistant function might be ameliorated.

Distinct expression and function of breast cancer metastasis suppressor 1 in mutant P53 glioblastoma.

Glioblastoma (GBM) is the most malignant subtype of astrocytic tumors with the worst prognosis in all its progressive forms. Breast cancer metastasis suppressor 1 (BRMS1) is a metastasis suppressor gene that controls malignancy in multiple tumors. As yet, however, its clinical and functional significance in mutant P53 GBM remains inconclusive. Here, we attempted to study the importance of BRMS1 in mutant P53 GBM. BRMS1 expression was evaluated in 74 human astrocytoma tissues by qRT-PCR, Western blotting and immunohistochemistry. BRMS1 expression in the astrocytoma tissues was correlated with clinicopathological parameters, the P53 mutation status and BRMS1 downstream targets, and compared with TCGA and NCI-60 datasets. siRNA-mediated knockdown of BRMS1 was performed in selected GBM cell lines to evaluate the functional role of BRMS1. Our study revealed an enhanced expression of BRMS1 in GBM which was associated with a poor patient survival, and this observation was corroborated by the TCGA dataset. We also found a positive correlation between BRMS1 expression and a mutant P53 status in GBM which was associated with a poor prognosis. In vitro BRMS1 silencing reduced the growth of mutant P53 GBM cells and repressed their colonization and migration/invasion by modulating EGFR-AKT/NF-κB signaling. Transcriptional profiling revealed a positive and negative correlation of uPA and ING4 expression with BRMS1 expression, respectively. Our data indicate upregulation of BRMS1 in high grade astrocytomas which correlates positively with mutant P53 and a poor patient survival. Silencing of BRMS1 in mutant P53 GBM cell lines resulted in a reduced cellular growth and migration/invasion by suppressing the EGFR-AKT/NF-kB signaling pathway. BRMS1 may serve as a predictive biomarker and therapeutic target in mutant P53 GBM.

Long Noncoding RNA LINC01554 Inhibits the Progression of NSCLC Progression by Functioning as a ceRNA for miR-1267 and Regulating ING3/Akt/mTOR Pathway.

Objectives This study focused on the biological functions and mechanisms of action of LINC01554 in nonsmall cell lung cancer (NSCLC). Methods The expression and prognostic values of LINC01554 in NSCLC were evaluated using The Cancer Genome Atlas datasets. MTT, colony formation, wound healing, transwell, and in vivo assays were performed to investigate the role of LINC01554 in NSCLC. The related protein expression levels were measured via western blotting. Bioinformatic analysis was conducted to predict targeted genes. The relationship between LINC01554, microRNA- (miR-) 1267, miR-1267, and inhibitor of growth family member 3 (ING3) was analysed via a dual-luciferase reporter assay. Results LINC01554 expression was downregulated in NSCLC and associated with NSCLC prognosis. LINC01554 overexpression suppressed NSCLC cell proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT). Bioinformatic and dual-luciferase reporter assays demonstrated that LINC01554 expression directly targeted miR-1267 expression, which in turn directly acted on ING3. An miR-1267 mimic significantly reduced ING3 expression, whereas an miR-1267 inhibitor observably elevated its expression. LINC01554 overexpression increased ING3 expression, whereas this effect was counteracted by the miR-1267 mimic. LINC01554 overexpression also significantly suppressed the expression of phosphorylated protein kinase B (Akt) and phosphorylated mammalian target of rapamycin (mTOR) expression; this effect was abrogated by the miR-1267 mimic. Mechanistically, LINC01554 overexpression repressed the growth, migration, invasion, and epithelial-mesenchymal transition (EMT) of NSCLC cells through the regulation of the miR-1267/ING3 axis via regulation of the Akt/mTOR signalling pathway. Conclusions We provide the first evidence of the involvement of the LINC01554/miR-1267 axis in NSCLC proliferation and metastasis through the ING3Akt/mTOR pathway. Thus, LINC01554 may serve as a novel therapeutic target for NSCLC.

Abstract 2426: GNG4 is a candidate G-proteinγ-subunit downstream of the Cxcl10/Cxcr3 axis that mediates cell migration in breast cancer

Abstract Introduction: Cxcl10 chemokine signaling has been implicated in cancer cell invasion and metastasis, but the mechanism is not well understood. Our prior studies have shown that deletion of the ING4 tumor suppressor in breast cancer cells enabled migration in response to Cxcl10. Further, migration was blocked by small molecule inhibition of either the Cxcl10 receptor Cxcr3, or Gβγ, but not Gαi, indicating that Gβγ was a key component downstream of Cxcl10/Cxcr3 signaling. There are 5 GNB genes encoding Gβ and 14 GNG genes encoding Gγ subunits in the human genome, however the precise subunits downstream of Cxcl10/Cxcr3 in breast cancer are unknown. Methods: RNA sequencing was used to identify differentially expressed genes in response to Cxcl10 treatment in ING4-deleted MDAmb-231 breast cancer cells, with a focus on GNB or GNG genes. Clinical and gene expression data of primary tumors obtained from NCBI GEO dataset GDS806 were used to evaluate correlation between GNG4 expression and patient survival. A CRISPR/Cas9 system was used to delete GNG4 in MDAmb231 cells and Western blot was used to confirm the gene deletion. We evaluated Cxcl10-induced migration of GNG4-deleted cells using transwell assays. Results: RNA sequencing showed that expression of GNG4 and GNG14 among the GNG genes was increased by 9 fold and 8 fold, respectively, in ING4-deleted MDAmb231 cells treated with Cxcl10, suggesting that these may encode effector(s) downstream of Cxc10/Cxcr3. GNG4 expression alone did not correlate with disease free survival (DFS) in breast cancer patients. However, a trend towards improved DFS was observed in patients with tumors expressing high ING4 and low GNG4 levels compared to those with low ING4 and high GNG4 expressing tumors (HR=0.32, 95% CI 0.10-1.03, p=0.051), suggesting high GNG4 expression in the absence of ING4 may contribute to aggressive breast cancer. Effects of GNG4 deletion in Cxcl10-induced migration are currently being evaluated. Conclusions: GNG4, encoding G-protein subunit γ-4, was induced following Cxcl10 treatment specifically in ING4-deleted breast cancer cells, suggesting that ING4 may repress GNG4 expression. Low ING4 and high GNG4 expression in breast tumors correlated with poor patient survival, implicating GNG4 as a candidate Gγ subunit in the Cxcl10/Cxcr3/ING4 axis. The function of GNG4 and GNG14 (Gγ-14) in Cxcl10-mediated breast cancer cell migration will be determined in the near future. These results may provide evidence that Gγ subunits, in particular, Gγ-4 or Gγ-14, play a role in Cxcl10/Cxcr3 signaling that mediates cancer cell migration. These results present Gβγ subunits as potential therapeutic targets for treatment of ING4-deficient breast cancer. Citation Format: Sukriti Bagchi, Suwon Kim. GNG4 is a candidate G-proteinγ-subunit downstream of the Cxcl10/Cxcr3 axis that mediates cell migration in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2426.

Transcriptional Regulation of ING5 and its Suppressive Effects on Gastric Cancer.

ING5 targets histone acetyltransferase or histone deacetylase complexes for local chromatin remodeling. Its transcriptional regulation and suppressive effects on gastric cancer remain elusive. Luciferase assay, EMSA, and ChIP were used to identify the cis-acting elements and trans-acting factors of the ING5 gene. We analyzed the effects of SAHA on the aggressive phenotypes of ING5 transfectants, and the effects of different ING5 mutants on aggressive phenotypes in SGC-7901 cells. Finally, we observed the effects of ING5 abrogation on gastric carcinogenesis. EMSA and ChIP showed that both SRF (−717 to −678 bp) and YY1 (−48 to 25bp) interacted with the promoter of ING5 and up-regulated ING5 expression in gastric cancer via SRF-YY1-ING5-p53 complex formation. ING5, SRF, and YY1 were overexpressed in gastric cancer, (P<0.05), and associated with worse prognosis of gastric cancer patients (P<0.05). ING5 had positive relationships with SRF and YY1 expression in gastric cancer (P<0.05). SAHA treatment caused early arrest at S phase in ING5 transfectants of SGC-7901 (P<0.05), and either 0.5 or 1.0 μM SAHA enhanced their migration and invasion (P<0.05). The wild-type and mutant ING5 transfectants showed lower viability and invasion than the control (P<0.05) with low CDC25, VEGF, and MMP-9 expression. Gastric spontaneous adenocarcinoma was observed in Atp4b-cre; ING5f/f, Pdx1-cre; ING5f/f, and K19-cre; ING5f/f mice. ING5 deletion increased the sensitivity of MNU-induced gastric carcinogenesis. ING5 mRNA might be a good marker of gastric carcinogenesis, and poor prognosis. ING5 expression was positively regulated by the interaction of SRF-YY1-ING5-p53 complex within the ING5 promoter from −50 bp upstream to the transcription start site. ING5 deletion might contribute to the tumorigenesis and histogenesis of gastric cancer.

Hsa_circ_0010729 is Involved in Oxygen-Glucose Deprivation/Reoxygenation-Induced Human Microvascular Endothelial Cell Deprivation by Targeting miR-665/ING5.

Ischemic stroke is a disease with high mortality. Circular RNA_0010729 (hsa_circ_0010729) has been reported to be involved in ischemic heart disease. However, it is not clear whether hsa_circ_0010729 is involved in the regulation of ischemic stroke. In this study, we used oxygen-glucose deprivation/reoxygenation (OGD/R) to stimulate human brain microvascular endothelial cells (HBMECs) model to investigate the potential role of hsa_circ_0010729 in stroke in vitro. The expression levels of hsa_circ_0010729, miR-665, and ING5 in ischemic stroke were detected by quantitative real-time polymerase chain reaction (qRT-PCR). HBMECs proliferation was detected by CCK-8. Cell apoptosis was detected by flow cytometry. The levels of inflammatory cytokines were detected by enzyme-linked immunosorbent assay (ELISA). Western blot was used to detect the related protein expression. Dual-luciferase reporter assay and RNA immunoprecipitation (RIP) were used to examine the target relationship between miR-665 and hsa_circ_0010729 or ING5. Compared with the control group, hsa_circ_0010729 and ING5 were highly expressed in OGD/R-induced HBMECs, while miR-665 was lowly expressed. Hsa_circ_0010729 silencing promoted OGD/R-induced cell proliferation and inhibited apoptosis. However, the effect of hsa_circ_0010729 down-regulation on OGD/R-induced cell was partially restored after co-transfection with miR-665 inhibitor. Overexpression of miR-665 can promote the proliferation and inhibit apoptosis of OGD/R-induced HBMECs by inhibiting ING5 expression. In OGD/R-induced HBMECs, hsa_circ_0010729 silencing decreased ING5 expression by upregulating miR-665. Hsa_circ_0010729 regulated miR-665/ING5 axis in OGD/R-induced HBMECs. Therefore, hsa_circ_0010729 may be a new therapeutic target for ischemic stroke.

A novel anti-inflammatory role links the CARS2 locus to protection from coronary artery disease

Background and aimsGenome-wide association studies (GWAS) identified a coronary artery disease (CAD) risk locus on 13.q34 tagged by rs61969072 (T/G). This variant lies in an intergenic region, proximal to ING1, CARKD and CARS2 but its causal relationship to CAD is unknown. Methods and ResultsWe first demonstrated that rs61969072 and tightly linked single nucleotide polymorphisms (SNPs) associate with CARS2 but not ING1 or CARKD expression in carotid endarterectomy samples, with reduced CARS2 abundance in carriers of the CAD risk allele (G). THP-1 monocytes were differentiated and polarized to proinflammatory (M1) and anti-inflammatory (M2) macrophages. CARS2 gene expression decreased in M1 and increased in M2 macrophages, consistent with a role for CARS2 in inflammation. Gene expression profiling revealed an increase in pro-inflammatory markers in response to CARS2 siRNA knockdown in THP-1 derived macrophages, accompanied by an increased abundance of inflammatory cytokines in the cell supernatant. Functional enrichment analysis of impacted transcripts identified the anti-inflammatory IL10 signalling pathway. Western blot analysis of CARS2 silenced macrophages revealed reduced STAT3 phosphorylation in response to IL-10 and increased expression of LPS-induced genes that are repressed by IL-10, indicating a role for CARS2 in anti-inflammatory signalling. Finally, to simulate vessel wall conditions, macrophages, and smooth muscle cells (SMC) were maintained in co-culture. Significantly, CARS2 silencing in macrophages altered the SMC phenotype, decreasing expression of contractile genes and increasing expression of inflammatory genes. ConclusionsThese data highlight a novel anti-inflammatory novel role for CARS2 in human macrophages and SMCs that may underlie the protective effect of a common GWAS-identified variant.

Abstract P5-09-04: Regulation of mTOR signaling pathway by ING4 in breast cancer: A new target in cancer therapy

Abstract Background: Breast cancer (BC) is one of the most diagnosed tumors in the United States. The mechanism of the disease development and progression is complex and still not fully understood. Dysregulation or change in the expression of transcription factors has been proposed as a possible cause of the disease severity and poor prognosis. The INhibitor of Growth family member 4 (ING4) is a tumor suppressor that has been connected to various cancer-related processes. Therefore, this study aims to assess the global gene regulation of BC cells by ING4 and identify the associated cellular processes and molecular pathways related to BC following ING4 loss. Methods: siRNA Knockdown and CRISPR Knockout technologies were used to deplete ING4 expression. Real-time PCR, RNA-Seq and Chip-Seq were used to identify gene expression alteration and ING4 enrichment on target promoters. Ingenuity Pathways Analysis (IPA®) was used to highlight candidates, networks and cellular processes. Results: Protein and RNA levels of ING4 were significantly reduced in MDA-231 knockdown and MCF7 knockout cells using siRNA and CRISPR technologies. RNA-Seq of BC cells revealed a large set of differentially expressed genes were regulated following ING4 loss. Key genes and biological networks related to cellular processes, including amino acid metabolism, cancer, cellular development, cellular growth and proliferation, were identified using IPA®. Chip-seq also revealed that ING4 was recruited to genes important in BC development and progression. Several genes were validated using the RT-qPCR method, and results were consistent with RNA-seq and Chip-seq data. Importantly, MDA-231 cells grown in stripped serum have shown an upregulation of the ribosomal protein S6 phosphorylation (PS6), an mTOR target protein, following ING4 knockdown. Treatment of MDA-231 cells with rapamycin blocked the phosphorylation of S6 induced by ING4 loss. An increase in S6 phosphorylation was also observed in MCF7 knockout cells grown either in regular or serum-free growth medium. The phosphorylation was reversed by amino acid transporter inhibitors JPH and BCH. Conclusion: The present study shows that ING4 regulates many genes involved in biological pathways and networks related to BC development and progression. Importantly, ING4 is a novel regulator of the mTOR pathway. Collectively, regulation of ING4 gene expression and its downstream molecules provides a novel approach in cancer treatment. Citation Format: Aymen Shatnawi, Travis Salisbury. Regulation of mTOR signaling pathway by ING4 in breast cancer: A new target in cancer therapy [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-09-04.

ING2 Controls Mitochondrial Respiration via Modulating MRPL12 Ubiquitination in Renal Tubular Epithelial Cells.

Mitochondrial injury of tubular epithelial cells (TECs) is the key pathogenic event underlying various kidney diseases and a potential intervening target as well. Our previous study demonstrated that ING2 is ubiquitously expressed at tubulointerstitial area within kidneys, while its role in regulating TEC mitochondrial respiration is not fully elucidated. To clarify the roles of ING2 in mitochondrial homeostasis of TECs and pathogenesis of acute ischemic kidney injury, Western blot, PCR, immunofluorescence, immunoprecipitation, and oxygen consumption rate assay were applied to address the roles of ING2 in modulating mitochondrial respiration. We further complemented these studies with acute ischemic kidney injury both in vitro and in vivo. In vitro study demonstrated ING2 could positively control TEC mitochondrial respiration. Concurrently, both mRNA and protein levels of mtDNA encoded respiratory chain components were altered by ING2, suggesting ING2 could regulate mtDNA transcription. In mechanism, ING2 could regulate the ubiquitination of a newly identified mitochondrial transcription factor MRPL12, thereby modulating its cellular stability and abundance. We also demonstrated ING2-mediated modulation on mtDNA transcription and mitochondrial respiration are involved in serum deprivation induced TEC injuries. Finally, immunohistochemistry study revealed that ING2 expression was significantly altered in kidney biopsies with acute ischemic kidney injury. In vivo study suggested that kidney specific ING2 overexpression could effectively ameliorate acute ischemic kidney injury. Our study demonstrated that ING2 is a crucial modulator of TEC mitochondrial respiration. These findings suggested a unrecognized role of ING2 in TEC mitochondrial energetic homeostasis and a potential intervening target for TEC mitochondrial injury associated pathologies.

Aberrant CREB1 activation in prostate cancer disrupts normal prostate luminal cell differentiation.

The molecular mechanisms of luminal cell differentiation are not understood well enough to determine how differentiation goes awry during oncogenesis. Using RNA-Seq analysis, we discovered that CREB1 plays a central role in maintaining new luminal cell survival and that oncogenesis dramatically changes the CREB1-induced transcriptome. CREB1 is active in luminal cells, but not basal cells. We identified ING4 and its E3 ligase, JFK, as CREB1 transcriptional targets in luminal cells. During luminal cell differentiation, transient induction of ING4 expression is followed by a peak in CREB1 activity, while JFK increases concomitantly with CREB1 activation. Transient expression of ING4 is required for luminal cell induction; however, failure to properly down-regulate ING4 leads to luminal cell death. Consequently, blocking CREB1 increased ING4 expression, suppressed JFK, and led to luminal cell death. Thus, CREB1 is responsible for the suppression of ING4 required for luminal cell survival and maintenance. Oncogenic transformation by suppressing PTEN resulted in constitutive activation of CREB1. However, the tumor cells could no longer fully differentiate into luminal cells, failed to express ING4, and displayed a unique CREB1 transcriptome. Blocking CREB1 in tumorigenic cells suppressed tumor growth in vivo, rescued ING4 expression, and restored luminal cell formation, but ultimately induced luminal cell death. IHC of primary prostate tumors demonstrated a strong correlation between loss of ING4 and loss of PTEN. This is the first study to define a molecular mechanism whereby oncogenic loss of PTEN, leading to aberrant CREB1 activation, suppresses ING4 expression causing disruption of luminal cell differentiation.

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.

ING4 Expression Landscape and Association With Clinicopathologic Characteristics in Breast Cancer

BackgroundBreast cancer (BC) development and progression is complex and still not fully understood. The expression or dysregulation of a variety of transcription factors has been suggested as contributing to disease severity and a poor prognosis. Therefore, the present study was designed to systematically outline ING4 expression and characteristics in clinical samples and cell lines of BC. Materials and MethodsA METABRIC (Molecular Taxonomy of Breast Cancer International Consortium) dataset was obtained from a cBioPortal public domain. ING4 gene expression, putative copy number alterations, and pertinent tumor information were retrieved. ING4 gene expression was identified for 1904 BC patients. ING4 mRNA expression data in BC cell lines were obtained from the Cancer Cell Line Encyclopedia. Analyses were conducted for associations between ING4 expression and age at diagnosis, tumor clinicopathologic characteristics, and molecular subtypes. The prognostic value of ING4 in BC patients was evaluated using Kaplan-Meier survival analysis. ResultsThe ING4 mRNA expression log intensity mean was 6.95, and 1005 (52.8%) of patients were determined to have high ING4 mRNA expression (mRNA log intensity > 6.95). Although ING4 gene expression correlated significantly and negatively with the Nottingham Prognostic Index and the number of positive lymph nodes (P < .05) and positively with overall survival time (P < .001). However, these correlations were weak in magnitude (r ∼ 0.1). The expression of ING4 was significantly associated with tumor grade, hormone receptor expression, human epidermal growth factor receptor 2, and molecular subtype. ING4 copy number alteration was also significantly associated with several clinicopathologic and prognostic factors. Kaplan-Meier survival analysis demonstrated greater overall survival for BC patients with high ING4 expression (P = .0046). Stratification of the data by menopausal status and tumor characteristics revealed a significant effect of ING4 expression on survival among premenopausal patients and women with a nonluminal subtype. Furthermore, the expression of ING4 in BC cell lines was significantly greater in luminal A and basal-like cells compared with human epidermal growth factor receptor 2-positive cells, which was also observed in the clinical samples. ConclusionsThe present study showed that ING4 gene expression is modulated in BC. High ING4 gene expression was associated with favorable prognostic parameters and positive clinical outcomes in our series of BC patients. ING4 could be used as a potential therapeutic target in BC-targeted therapy.

Ing4 Deficiency Reprograms Multipotent Progenitor Cells to Confer Self-Renewal Capabilities

A network of transcription factors and associated complexes regulate the process of hematopoiesis and are required for maintenance and development of the hematopoietic program. Ing4, a tumor suppressor protein, was identified in a screen for epigenetic regulators of hematopoiesis in zebrafish as required for specification of hematopoietic stem and progenitor cells (HSPCs). Recent work has shown that Ing4 is inactivated in various cancer cells. This inactivation promotes stem cell-like qualities in malignant cells. Ing4 plays an inhibitory role in the NF-κB pathway, conferring, in part, Ing4's tumor-suppressor capability. Loss of Ing4 is correlated to diminished hematopoietic stem cell (HSC) specification in zebrafish and increased NF-κB target gene expression. NF-κB knockdown assays in zebrafish embryos suggest inhibition of NF-κB remediates loss of Ing4 expression, with HSC rescue efficacy varying directly with concentration of inhibitor. Similarly, the necessity of Ing4 in murine hematopoiesis has been observed. Here, Ing4 deficiency impairs HSC function, while simultaneously enhancing the regenerative capacity of multipotent progenitor cells (MPPs). Characterization of bone marrow from Ing4-deficient mice shows abnormal hematopoiesis, with a striking decrease in MPPs as compared to wildtype mice (47.9% vs 19.3%). Hematopoiesis under stress conditions is also altered in Ing4-deficient mice, as observed following competitive HSC transplantation. In a surprising finding, MPPs from Ing4-deficient mice showed a dramatic increase in peripheral blood multilineage chimerism compared to wildtype mice up to 9 months post-transplantation in a competitive transplant assay (19% vs. 59%). This supports the hypothesis that MPPs from Ing4-deficient mice have enhanced self-renewal capacity. Additionally, we have observed a subpopulation of Ing4-deficient MPPs that express lower levels of CD34, CD34+/mid. This population of CD34+/mid cells was also shown to express CD229 (85% positive), while very few WT MPPs express both CD34+/mid and CD229 (5.0%). Reduced levels of CD34 expression combined with CD299 are known to be markers of HSCs, and so we hypothesize that a subset of CD34+/midCD229+ MPPs in Ing4-deficient mice retain their self-renewal capacity. Taken together, our data suggest Ing4 typically functions as a suppressor of genes necessary for self-renewal and developmental potency of MPPs. Additionally, cell cycle analysis combined with Ki-67 expression showed Ing4-deficient MPPs have enhanced ability to maintain quiescence, with 15.2% of cells found to be in G0 phase as compared to 6.5% of wildtype MPPs in G0. Finally, after 5-FU treatment, levels of MPPs in WT mice were similar pre- and post-treatment. Future experiments will seek to elucidate this observation in consideration of the pro-inflammatory environment. These findings suggest Ing4 is a critical regulator of hematopoiesis, and these data provide important clues for further characterization of the pathways and functions of Ing4. Our data show that Ing4 deficiency promotes stem cell-like properties in MPPs, suggesting it has crucial regulatory functions in both stem cell self-renewal and maintenance. Disclosures No relevant conflicts of interest to declare.

Effect of bevacizumab on expression level of GLI1 and ING4 in colon cancer animal model.

This study aimed to investigate the effect of bevacizumab on GLI1 and ING4 expression in colon cancer animal model. Colon cancer model in rats was induced by azoxymethane (AOM). Bevacizumab was used for the treatment of colon cancer rats. Tumor volume and weight were measured, tumor growth curve was visualized and tumor inhibition rate was calculated. GLI1 and ING4 of colon cancer cells were silencing expressed. Western blot analysis was used to detect the expressions of GLI1, ING4, caspase-3, Bax, β-catenin, Bcl2, PTEN, PI3K, Akt, NF-κB. The apoptosis rate was detected by flow cytometry. MTT assay was used to detect cell activity to get IC50 value. After AOM induced colon cancer model in rats, the expressions of ING4, caspase-3, Bax and PTEN were downregulated, the expressions of GLI1, β-catenin, Bcl2, PI3K, Akt and NF-κB were upregulated and the apoptosis rate was downregulated. After bevacizumab treatment, the tumor volume and weight decreased, the expressions of ING4, caspase-3, Bax, PTEN were upregulated, the expressions of GLI1, β-catenin, Bcl2, PI3K, Akt, NF-κB were downregulated, and the cell apoptosis rate increased. Cell experiments showed that GLI1 promotes tumor growth and reduces the sensitivity of bevacizumab, while ING4 inhibits tumor growth and increases the sensitivity of bevacizumab. Bevacizumab inhibits the growth of colon cancer tumor by upregulating ING4 and downregulating GLI1.

Abstract B06: ING2 loss sensitizes KRAS-mutated NSCLC to WEE1 inhibition through regulation of CHK1 expression

Abstract ING2 (Inhibitor of Growth 2) is a tumor-suppressor gene involved in chromatin acetylation whose loss has been observed in nearly 60% of NSCLC. The loss of ING2 expression has been associated with the progression of tumor cells from G1 to S phase, replication stress and genomic instability. The intra-S and the G2-M phase checkpoints are mainly regulated by the cell cycle tyrosine kinase WEE1. Therefore, we hypothesized that the loss of ING2 expression could sensitize NSCLC to the WEE1 inhibitor MK-1175, according to a synthetic lethality model. In silico analysis of the cancer gene dependency map (shRNAi Achilles library v.2016) was used to explore gene interactions with ING2. Antiproliferative effects of drugs were tested in vitro with AlamarBlue and MTT assays in a panel of NSCLC cells lines with varied mutational status. Multiple functional approaches were taken to decrease ING2 expression in RAS-mutated NSCLC cell lines (si-RNA, LNA-gapmer, and CrispR-Cas9). Pharmacologic inhibition of ING2 epigenetic functions was achieved using the HDAC inhibitor SAHA. Cell cycle and apoptosis analysis were performed using flow cytometry according to a BrdU-Pi and Annexin V-PI protocols, respectively. Compusyn software was used to model MK-1775 and SAHA drug interaction. NSCLC cell lines with mutant RAS that expressed high levels of ING2 protein were more resistant to the antiproliferative effect of MK-1775 than similar lines with low levels of ING2. Colony formation assays were consistent with these results. Multiple functional assays reproducibly showed that downregulation of ING2 expression increased MK-1775 sensitivity in vitro. Similar results were obtained with pharmacologic disruption of ING2 from the HDAC-SIN3A complex using SAHA, where disruption conferred a higher antiproliferative effect to MK-1775. Interestingly, combination treatment induced cell cycle progression and synergic cell death through apoptosis in RAS-mutated cell lines nondeficient for ING2. Since the synergistic effect of SAHA and WEE1 inhibitor was shown to be dependent on CHK1 activity in a previous report, we checked whether ING2 and CHK1 expression could be linked. Using public database and functional assays, we indeed find that ING2 downregulation represses CHK1 transcription through a mechanism that remains to be elucidated. Collectively, these findings demonstrate that inhibition of WEE1 by MK-1775 could represent a therapeutic approach to more effectively target ING2-deficient RAS-mutated NSCLC. Moreover, HDAC inhibitors targeting ING2 epigenetic functions synergize with MK-1775, possibly through regulation of CHK1 transcription. Consequently, this combination therapy could also be a promising strategy in ING2-proficient RAS-mutated NSCLC. Citation Format: Charles Ricordel, Subash Thalappilly, Jérome Archambeau, Angela Chan, Nancy Nixon, Benoît Desrues, Gwyn Bebb, Karl Riabowol, Rémy Pedeux. ING2 loss sensitizes KRAS-mutated NSCLC to WEE1 inhibition through regulation of CHK1 expression [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr B06.

High level of H3K4 tri-methylation modification predicts poor prognosis in esophageal cancer.

Objectives: An increase in the trimethylation of lysine 4 of histone 3 (H3K4me3) has been reported to be involved in the development of several types of tumors. However, the level and role of H3K4me3 in human esophageal cancer (HEC) remain unknown. Here, we assessed the role and clinical significance of H3K4me3 in HEC.Methods: The level of H3K4me3 was determined in 15 pairs of HEC and paracancerous tissues by Western blotting. A tissue microarray including samples from 100 HEC patients was analyzed by immunohistochemistry to determine the relationship between the level of H3K4me3 and the clinicopathological features of HEC patients. Then, the levels of H3K4me3 in HEC cells were elevated via knockdown of inhibitor of growth family member 4(Ing4) expression. Finally, the prognostic significance of H3K4me3 levels in HEC patients was further analyzed.Results: We found that H3K4me3 levels were frequently elevated in HEC tissues compared with adjacent esophageal tissues, and elevated H3K4me3 was significantly associated with poor tumor differentiation (p =1.39×10-5) and advanced tumor stage (p=8.5×10-5). After Ing4 knockdown in HEC cells, we found that the cell proliferation, metastasis, invasion and colony formation abilities were enhanced compared to those in the control cells. Notably, we found that HEC patients with a high level of H3K4me3 exhibited an unfavorable 5-year survival rate compared to those with a low level of H3K4me3 (p=6.8×10-5). The univariate analysis showed that the tumor differentiation, TNM stage, and H3K4me3 level were predictors of the overall survival rate of HEC patients. In the multivariate analysis, tumor stage (p=0.015) and H3K4me3 level (p=0.034) were revealed to be independent parameters for predicting the prognosis of HEC patients.Conclusions: Thus, high levels of H3K4me3 may be used as a meaningful biomarker for HEC prognosis evaluation.