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Here, we found that ING5 overexpression suppressed cell viability, glucose metabolism, migration, invasion and epithelial-mesenchymal transition, and induced cell arrest, apoptosis, senescence, autophagy and fat accumulation in ovarian cancer cells. ING5-mediated chemoresistance was positively linked to apoptotic resistance and chemoresistance-related gene expression. ING5 overexpression suppressed tumor growth of ovarian cancer by decreasing proliferation, and inducing apoptosis and autophagy. ING5 mRNA level was lower in ovarian cancer than normal ovary, and borderline than benign tumors ( p < 0.05), and negatively correlated with vascular invasion, lymphatic invasion and FIGO staging of ovarian cancer ( p < 0.05). ING5 protein was less expressed in primary cancer than normal ovary ( p < 0.05). There was a negative correlation between ING5 mRNA expression and the overall or progression-free survival time of the cancer patients with Grade 2, Grade 3, and stage I cancer ( p < 0.05). Immunohistochemically, ING5 was less expressed in serous and mucinous adenocarcinoma than miscellaneous subtypes, and positively correlated with dedifferentiation and ki-67 expression of ovarian cancer ( p < 0.05). These data suggested that down-regulated ING5 expression might be involved in ovarian carcinogenesis possibly by suppressing aggressive phenotypes, including proliferation, tumor growth, migration, invasion, and anti-apoptosis., Competing Interests: CONFLICTS OF INTEREST The authors have declared that no competing interests exist.

Here, we found that ING5 overexpression resulted in a lower proliferation, reduced glucose metabolism, S arrest, decreased migration and invasion, apoptotic induction, fat accumulation, autophagy, senescence and mesenchymal-epithelial-transition of breast cancer cells. It also suppressed the tumor growth of breast cancer cells by inhibiting proliferation, inducing apoptosis and autophagy. ING5-mediated chemoresistance was positively linked to Akt and NF-κB activation, MRP1 and GST-π overexpression, and FBXW7 hypoexpression. ING5 expression was higher in breast cancer than normal tissue at both mRNA and protein levels. ING5 mRNA expression was positively correlated with relapse- and distant metastasis-free survival rates. Nuclear ING5 expression showed gradual decrease from breast normal tissue, fibroadenoma, adenomatosis, primary to metastatic cancers, while versa for cytoplasmic ING5. Nuclear ING5 expression was negatively correlated with distant metastasis and p53 hypoexpression, while cytoplasmic ING5 expression was positively correlated with tumor size and ER expression. These data suggested that up-regulated expression and nucleocytoplasmic translocation of ING5 protein were observed in breast cancer. The higher expression of nuclear ING5 was inversely linked to worse clinicopathological behaviors of breast cancer by in vivo and vitro reversing aggressive phenotypes. Therefore, it should be employed as a biomarker to indicate the tumorigenesis and aggressiveness of breast cancer, and as a potential target for gene therapy., Competing Interests: CONFLICTS OF INTEREST The authors have declared that no competing interests exist.

To elucidate the anti-tumor effects and molecular mechanisms of ING5 on glioma cells, we overexpressed it in U87 cells, and examined the phenotypes and their relevant molecules. It was found that ING5 overexpression suppressed proliferation, energy metabolism, migration, invasion, and induced G 2 /M arrest, apoptosis, dedifferentiation, senescence, mesenchymal- epithelial transition and chemoresistance to cisplatin, MG132, paclitaxel and SAHA in U87 cells. There appeared a lower expression of N-cadherin, Twist, Slug, Zeb1, Zeb2, Snail, Ac-H3, Ac-H4, Cdc2, Cdk4 and XIAP, but a higher expression of Claudin 1, Histones 3 and 4, p21, p53, Bax, β-catenin, PI 3 K, Akt, and p-Akt in ING5 transfectants. ING5 overexpression suppressed tumor growth of U87 cells in nude mice by inhibiting proliferation and inducing apoptosis. Down-regulated ING5 expression was closely linked to the tumorigenesis and histogenesis of glioma. These data indicated that ING5 expression might be considered as a good marker for the tumorigenesis and histogenesis of gliomas. It might be employed as a potential target for gene therapy of glioma. PI 3 K/Akt or β-catenin/TCF-4 activation might be positively linked to chemotherapeutic resistance, mediated by ING5., Competing Interests: CONFLICTS OF INTEREST The authors declare that they have no competing interests.

ING5 belongs to the Inhibitor of Growth (ING) candidate tumor suppressor family, whose functions have been involved in the regulation of chromatin remodeling, cell cycle progression, proliferation and apoptosis. Our previous study has shown that ING5 overexpression inhibits lung cancer aggressiveness via suppressing epithelial to mesenchymal transition (EMT). However, the mechanisms remain largely unknown. In the current study, by Phospho-Kinase array and western blot, we have defined significantly upregulated EGFR/PI3K/Akt and IL-6/STAT3 oncogenic signaling pathways in ING5 knockdown A549 cells, which could be downregulated by ING5 overexpression. PI3K inhibitor ZSTK474 or STAT3 inhibitor Niclosamide not only abolished ING5 knockdown-promoted proliferation, colony formation, migration and invasion of lung cancer A549 cells, but also impaired ING5 knockdown-stimulated metastasis of cancer cells in mouse xenograft models with tail vein injection of A549 cells. Furthermore, treatment with ZSTK474 or Niclosamide decreased protein level of EGFR, p-Akt, IL-6 and p-STAT3, and reversed ING5 knockdown-promoted EMT, as indicated by downregulated expression of EMT marker E-cadherin, an epithelial marker, increased expression of N-cadherin, a mesenchymal marker, and EMT-related transcription factors including Snail, Slug, Smad3 and Twist. Taken together, these results demonstrate that loss of ING5 enhances aggressiveness of lung cancer cells by promoting EMT via activation of EGFR/PI3K/Akt and IL-6/STAT3 signaling pathways., Competing Interests: CONFLICTS OF INTEREST The authors declare that they have no competing interests.

ING5 can interact with p53, thereby inhibiting cell growth and inducing apoptosis. We found that ING5 overexpression not only inhibited proliferation, migration, and invasion, but also induced G2 arrest, differentiation, autophagy, apoptosis, glycolysis and mitochondrial respiration in lung cancer cells. ING5 transfection up-regulated the expression of Cdc2, ATG13, ATG14, Beclin-1, LC-3B, AIF, cytochrome c, Akt1/2/3, ADFP, PFK-1 and PDPc, while down-regulated the expression of Bcl-2, XIAP, survivin,β-catenin and HXK1. ING5 transfection desensitized cells to the chemotherapy of MG132, paclitaxel, and SAHA, which paralleled with apoptotic alteration. ING5 overexpression suppressed the xenograft tumor growth by inhibiting proliferation and inducing apoptosis. ING5 expression level was significantly higher in normal tissue than that in lung cancer at both protein and mRNA levels. Nuclear ING5 expression was positively correlated with ki-67 expression and cytoplasmic ING5 expression. Cytoplasmic ING5 expression was positively associated with lymph node metastasis, and negatively with age, lymphatic invasion or CPP32 expression. ING5 expression was different in histological classification: squamous cell carcinoma > adenocarcinoma > large cell carcinoma > small cell carcinoma. Taken together, our data suggested that ING5 downregulation might involved in carcinogenesis, growth, and invasion of lung cancer and could be considered as a promising marker to gauge the aggressiveness of lung cancer. It might be employed as a potential target for gene therapy of lung cancer.

Unlabelled: Hepatitis B virus (HBV) is a major risk factor for development and progression of hepatocellular carcinoma (HCC). It has been reported that viral infection can interfere with cellular microRNA (miRNA) expression and participate in the pathogenesis of oncogenicity. Our miRNAs array data indicated that miR-331-3p expression in HCC cell lines increased, but the relationship between miR-331-3p expression and HBV activity is unclear. Here, we observed elevated expression of miR-331-3p in different HCC cell lines expressing HBV. HBV, especially HBx, promotes miR-331-3p expression by enhancing its promoter activity. Using a miRNA target prediction database miRBase, we identified ING5 to be a novel target gene of miR-331-3p. miR-331-3p could inhibit ING5 expression by directly targeting its 3'-untranslated region (3'-UTR). As predicted, HBV was confirmed to repress ING5 at both mRNA and protein levels by promoting miR-331-3p expression. Our result indicated that miR-331-3p expression promotes proliferation of SMMC7721 cells by inhibiting ING5. ING5 overexpression promoted cell apoptosis in HCC cell lines. We also found ING5 expression was decreased in tumor tissue of HCC patient with HBV infection compared to its expression in para-carcinoma tissues., Conclusion: These results showed that miR-331-3p is upregulated by HBV and promotes proliferation of HCC cells though repression of ING5 expression. These data provide new insights for understanding the mechanisms of HBV-related HCC pathogenesis.

Here, we found that ING5 overexpression increased autophagy, differentiation, and decreased proliferation, apoptosis, migration, invasion and lamellipodia formation in gastric cancer cells, while ING5 knockdown had the opposite effects. In SGC-7901 transfectants, ING5 overexpression caused G1 arrest, which was positively associated with 14-3-3 overexpression, Cdk4 and c-jun hypoexpression. The induction of Bax hypoexpression, Bcl-2, survivin, 14-3-3, PI3K, p-Akt and p70S6K overexpression by ING5 decreased apoptosis in SGC-7901 cells. The hypoexpression of MMP-9, MAP1B and flotillin 2 contributed to the inhibitory effects of ING5 on migration and invasion of SGC-7901 cells. ING5 overexpression might activate both β-catenin and NF-κB pathways in SGC-7901 cells, and promote the expression of down-stream genes (c-myc, VEGF, Cyclin D1, survivin, and interleukins). Compared with the control, ING5 transfectants displayed drug resistance to triciribine, paclitaxel, cisplatin, SAHA, MG132 and parthenolide, which was positively related to their apoptotic induction and the overexpression of chemoresistance-related genes (MDR1, GRP78, GRP94, IRE, CD147, FBXW7, TOP1, TOP2, MLH1, MRP1, BRCP1 and GST-π). ING5 expression was higher in gastric cancer than matched mucosa. It was inversely associated with tumor size, dedifferentiation, lymph node metastasis and clinicopathological staging of cancer. ING5 overexpression suppressed growth, blood supply and lung metastasis of SGC-7901 cells by inhibiting proliferation, enhancing autophagy and apoptosis in xenograft models. It was suggested that ING5 expression might be employed as a good marker for gastric carcinogenesis and subsequent progression by inhibiting proliferation, growth, migration, invasion and metastasis. ING5 might induce apoptotic and chemotherapeutic resistances of gastric cancer cells by activating β-catenin, NF-κB and Akt pathways.

The proteins of the Inhibitor of Growth (ING) candidate tumor suppressor family are involved in multiple cellular functions such as cell cycle regulation, apoptosis, and chromatin remodeling. ING5 is the new member of the family whose actual role in tumor suppression is not known. Here we show that ING5 overexpression in lung cancer A549 cells inhibited cell proliferation and invasiveness, while ING5 knockdown in lung cancer H1299 cells promoted cell aggressiveness. ING5 overexpression also abrogated tumor growth and invasive abilities of lung cancer cells in mouse xenograft models. Further study showed that ING5 overexpression inhibited EMT indicated by increase of E-cadherin and decrease of N-cadherin, Snail and slug at mRNA and protein levels, which was accompanied with morphological changes. cDNA microarray and subsequent qRT-PCR validation revealed that ING5 significantly downregulated expression of EMT (epithelial to mesenchymal transition)-inducing genes including CEACAM6, BMP2 and CDH11. Clinical study by tissue microarray showed that nuclear ING5 negatively correlated with clinical stages and lymph node metastasis of lung cancer. Furthermore, high level of nuclear ING5 was associated with a better prognosis. Taken together, these findings uncover an important role for ING5 as a potent tumor suppressor in lung cancer growth and metastasis.

As the major barrier to curative cancer chemotherapy, chemoresistance presents a formidable challenge to both cancer researchers and clinicians. We have previously shown that the bladder cancer (BCa) cell line 5637 is significantly more sensitive to the cytoxicity of five chemotherapeutic agents than H-bc cells. Using an RNA-seq-based omic analysis and validation at both the mRNA and protein levels, we found that the inhibitor of growth 5 (ING5) gene was upregulated in 5637 cells compared with H-bc cells, indicating that it has an inhibitory role in BCa chemoresistance. siRNA-mediated inhibition of ING5 increased the chemoresistance and inhibited the DNA damage response pathway in 5637 cells. Conversely, forced expression of EGFP-ING5 decreased the chemoresistance of and activated the DNA damage response pathway in H-bc cells. We also showed that ING5 gene expression is inhibited by miR-193a-3p and is instrumental in miR-193a-3p's role in activating BCa chemoresistance. Our results demonstrate both the role and mechanism of inhibition of BCa chemoresistance by ING5.