Macrophages are essential inflammatory cells which regulate the features of immune reactions within tumors. Many studies have reported their regulatory roles in immunity through cytokines and cell signaling. However, relatively few studies have focused on their metabolic features and mechanisms. We aimed to determine the signaling pathway regulating cell metabolism and the mechanism related to the regulation of human tumor-associated macrophages (TAMs) in gastric cancer (GC). Tumor-infiltrated macrophages were isolated from human GC tissues using magnetic beads, gene transcription was determined by real-time PCR, protein expression was monitored using western blots, metabolites were determined using HPLC, and transcriptional regulation was analyzed by the luciferase-based reporter gene system. A significant decrease in microRNA (miR)-30c and an increase in regulated in development and DNA damage responses 1 (REDD1) were detected in human GC TAMs, the transcription of miR-30c was negatively correlated with REDD1. MicroRNA-30c expression was suppressed by hypoxia-inducible factor-1α activation and related to decreased mTOR activity as well as glycolysis in human GC TAMs. Hypoxia-regulated miR-30c downregulated REDD-1 expression by targeting its 3'UTR. Overexpression of miR-30c or restored mTOR activity in macrophages with miR-30c

Methamphetamine (METH) is a widely abused illicit psychoactive drug. Our previous study has shown that CCAAT-enhancer binding protein β (C/EBPβ) is an important regulator in METH-induced neuronal autophagy and apoptosis. However, the detailed molecular mechanisms underlying this process remain poorly understood. Previous studies have demonstrated that DNA damage-inducible transcript 4 (DDIT4), Trib3 (tribbles pseudo kinase 3), alpha-synuclein (α-syn) are involved in METH-induced dopaminergic neurotoxicity. We hypothesized that C/EBPβ is involved in METH-induced DDIT4-mediated neuronal autophagy and Trib3-mediated neuronal apoptosis. We tested our hypothesis by examining the effects of silencing C/EBPβ, DDIT4, Trib3 or α-syn with small interfering ribonucleic acid (siRNA) on METH-induced autophagy and apoptosis in the human neuroblastoma SH-SY5Y cells. We also measured the levels of phosphorylated tuberous sclerosis complex 2 (TSC2) protein and Parkin protein level in SH-SY5Y cells. Furthermore, we demonstrated the effect of silencing C/EBPβ on METH-caused neurotoxicity in the striatum of rats by injecting LV-shC/EBPβ lentivirus using a stereotaxic positioning system. The results showed that METH exposure increased C/EBPβ, DDIT4 protein expression. Elevated DDIT4 expression raised up p-TSC2/TSC2 protein expression ratio, inhibited mTOR signaling pathway, activating cell autophagy. We also found that METH exposure increased the expression of Trib3, α-syn, decreased the Parkin protein expression. Lowering levels of Parkin raised up α-syn expression, which initiated mitochondrial apoptosis by down-regulating anti-apoptotic Bcl-2, followed by up-regulation of pro-apoptotic Bax, resulting in translocation of cytochrome c (cyto c), an apoptogenic factor, from the mitochondria to cytoplasm and activation of caspase-dependent pathways. These findings were supported by data showing METH-induced autophagy and apoptosis was significantly inhibited by silencing C/EBPβ, DDIT4, Trib3 or α-syn, or by Parkin over-expression. Based on the present data, a novel of mechanism on METH-induced cell toxicity is proposed, METH exposure increased C/EBPβ protein expression, triggered DDIT4/TSC2/mTOR signaling pathway, and evoked Trib3/Parkin/α-syn-related mitochondrial apoptotic signaling pathway. Collectively, these results suggest that C/EBPβ plays an important role in METH-triggered autophagy and apoptosis and it may be a potential target for therapeutics in METH-caused neurotoxicity.

BACKGROUND: Regulated in development and DNA damage response (REDD1), a gene responding to hypoxia or multiple DNA damage events, was recently implicated in cancer development and progression. Previously, in vivo and in vitro experiments indicated that REDD1 functions as an oncogene in ovarian cancer cells. However, the role of REDD1 in cancer cell migration and invasion and in clinical significance of prognostic values is not examined in detail.
METHODS: We detected the REDD1 protein expression by immunohistochemistry in 18 normal ovarian surface epithelium or fallopian tube epithelium specimens, 24 ovarian borderline tumors, and 229 ovarian cancers. Fisher's exact test, logistic regression analysis, the Kaplan-Meier method, and the log-rank test were used to evaluate the association of REDD1 with clinical factors, overall survival and disease-free survival. The prognostic predictive value of REDD1 for ovarian cancer patients was evaluated using multivariate Cox proportional hazard regression models. REDD1 expression in HEY, HEY A8, SKOV3, SKOV3 ip1, OVCA429, OVCA433 and A2780 human ovarian epithelial cancer cell lines was detected by western blotting. The role of REDD1 in cell invasion and migration was assessed by transwell migration and invasion assays using SKOV3, A2780, HEY, HEYA8, and SKOV3-REDD1 with parental A2780-REDD1 HEY-REDD1i and HEY A8-REDD1i.
RESULTS: High expression of REDD1 was observed in 35.4% of primary ovarian carcinoma samples. Overexpression of cytoplasmic REDD1 in ovarian cancer was significantly associated with serous carcinoma (P < 0.001), late-stage disease (P < 0.001), ascites (P < 0.001), and partial or non-response to chemotherapy (P < 0.001). High cytoplasmic expression of REDD1 was correlated with poorer overall survival (P < 0.001) and disease-free survival (P < 0.001). The multivariate Cox proportional hazards regression analysis indicated that patients with high cytoplasmic REDD1 expression had a high risk of death (P < 0.001) and high risk of an event (i.e., recurrence, progression, or death) (P < 0.001). REDD1 was first reported as an independent prognostic factor in ovarian cancer patients. In addition, REDD1 overexpression enhanced ovarian cancer cell migration and invasion.
CONCLUSION: REDD1 is an independent unfavorable prognostic factor in ovarian carcinoma and may promote ovarian cancer metastasis.

BACKGROUND/AIMS: Radix Angelica Sinensis (danggui in Chinese) is widely used in traditional chinese medicine (TCM). N-butylidenephthalide (BP), a bioactive compound in danggui, is a potential antitumor agent for various cancer types. However, its clinical effect and mechanism in the treatment of gastric cancer remain undetermined.
METHODS: The in vivo protective effect of danggui in patients with gastric cancer were validated using data from Taiwan's National Health Insurance Research Database (NHIRD). The genes induced by BP-treatment were analyzed by whole transcriptome RNA sequencing (RNA-seq) and validated by real-time PCR, western blot and siRNA transfection. The effect of BP on AGS cell migration and invasion was evaluated in transwell assays. The antitumor effects of BP were evaluated in vivo in an AGS xenograft animal model.
RESULTS: Danggui users were found to have an increased survival rate when compared with danggui nonusers (log-rank test p = 0.002) . The use of danggui highly associated with decreased mortality (the adjusted hazard ratio (HR) of danggui user was 0.72 [95 % CI, 0.57-0.92] (p = 0.009). The in vitro results showed that BP inhibited gastric cancer cell proliferation, and triggered cellular apoptosis depending on the activation of mitochondrial apoptotic pathway. Using RNA-seq analysis we found that REDD1 was the highest transcript induced by BP in gastric cancer cells. BP induce an increase of REDD1 expression that inhibits mTOR signaling, thus inhibiting gastric cancer growth. We used RNA interference to demonstrate that the knock-down of REDD1 attenuated the BP-induced mTORC1 activation and growth inhibition. BP suppressed the growth of AGS xenografts tumor in vivo.
CONCLUSION: Danggui can prolong the survival rate of gastric cancer patients in Taiwan. BP caused gastric cancer cell death through the activation of mitochondria-intrinsic pathway and induced the REDD1 expression leading to mTOR signal pathway inhibition in gastric cancer cells. BP inhibited the in vivo growth of AGS xenograft tumors. These results may provide the basis for a new therapeutic approach toward the treatment of gastric cancer progression.

BACKGROUND: Gastric cancer (GC) is one of the most common malignancies worldwide, particularly in China. DNA damage-inducible transcript 4 (DDIT4) is a mammalian target of rapamycin inhibitor and is induced by various cellular stresses; however, its critical role in GC remains poorly understood. The present study aimed to investigate the potential relationship and the underlying mechanism between DDIT4 and GC development.
METHODS: We used western blotting, real-time polymerase chain reaction, and immunohistochemical or immunofluorescence to determine DDIT4 expression in GC cells and tissues. High-content screening, cell counting kit-8 assays, colony formation, and in vivo tumorigenesis assays were performed to evaluate cell proliferation. Flow cytometry was used to investigate cell apoptosis and cell cycle distribution.
RESULTS: DDIT4 was upregulated in GC cells and tissue. Furthermore, downregulating DDIT4 in GC cells inhibited proliferation both in vitro and in vivo and increased 5-fluorouracil-induced apoptosis and cell cycle arrest. In contrast, ectopic expression of DDIT4 in normal gastric epithelial cells promoted proliferation and attenuated chemosensitivity. Further analysis indicated that the mitogen-activated protein kinase and p53 signaling pathways were involved in the suppression of proliferation, and increased chemosensitivity upon DDIT4 downregulation.
CONCLUSION: DDIT4 promotes GC proliferation and tumorigenesis, providing new insights into the role of DDIT4 in the tumorigenesis of human GC.

Acute myeloid leukemia (AML) is the most common type of acute leukemia and is a heterogeneous clonal disorder. At present, the pathogenesis of AML and potential methods to effectively prevent AML have become areas of interest in research. In the present study, two messenger ribonucleic acid sequencing datasets of patients with AML were downloaded from the Cancer Genome Atlas and Gene Expression Omnibus databases. The differentially expressed genes (DEGs) of the poor and good prognosis groups were screened using the Linear Models for Microarray Data package, and the prognosis‑related genes were screened using univariate Cox regression analysis. A total of 206 significant DEGs were identified. Following univariate and multivariate Cox regression analysis, 14 genes significantly associated with prognosis were screened and six of these genes, including triggering receptor expressed on myeloid cells 2 (TREML2), cysteine‑glutamate transporter (SLC7A11), NACHT, LRR, and PYD domains‑containing protein 2 (NLRP2), DNA damage‑inducible transcript 4 protein (DDIT4), lymphocyte‑specific protein 1 (LSP1) and C‑type lectin domain family 11 member A (CLEC11A), were used to construct model equations for risk assessment. The prognostic scoring system was used to evaluate risk for each patient, and the results showed that patients in the low‑risk group had a longer survival time, compared with those in the high‑risk group (P=9.59e‑06 for the training dataset and P=0.00543 for the validation dataset). A total of eight main Kyoto Encyclopedia of Genes and Genomes pathways were identified, the top three of which were hematopoietic cell lineage, focal adhesion, and regulation of actin cytoskeleton. Taken together, the results showed that the scoring system established in the present study was credible and that the six genes were identified, which were significantly associated with the risk assessment of AML, offer potential as prognostic biomarkers. These findings may provide clues for further clarifying the pathogenesis of AML.

PURPOSE: Rac3 plays an important role in regulating tumorigenesis. Autophagy plays a vital role in tumorigenesis and tumor progression. The relationship between the two remains unclear. The objective of the present study was to determine the specific molecular mechanism of intracellular Rac3 in regulating autophagy and reveal the relationship between tumor cell autophagy and apoptosis.
METHODS: A laser confocal microscope was used to photograph the accumulated EGFP-MAP1LC3 spots for investigating the relationship between Rac3 and autophagy at the cellular level. Immunoblotting was also used to investigate the relationship between Rac3 and autophagy. The autophagy flux arising from inhibition of Rac3 was detected with autophagy inhibitors and ATG5 and ATG7 siRNA interference experiments. ATF4 and DDIT4 siRNA interference and overexpression experiments were conducted to investigate the relationship between endoplasmic reticulum stress, the MTOR signaling pathway, and autophagy arising from inhibition of Rac3. Co-immunoprecipitation experiments were performed to investigate the interaction between Rac3 and proteins related to endoplasmic reticulum stress. Co-immunoprecipitation was performed to investigate the structural domains between Rac3 and HSPA5.
RESULTS: The expression of ATF4 and DDIT4 was upregulated, which inhibited the MTOR signaling pathway and induced autophagy of human non-small cell lung cancer cells after Rac3 siRNA was introduced. The degree of acetylation of the substrate, HSPA5, increased and the endoplasmic reticulum stress response was activated after Rac3 was inhibited.
CONCLUSION: In conclusion, the degree of acetylation of HSPA5 increased and it was dissociated from the receptor, EIF2AK3, on the endoplasmic reticulum membrane, thus causing the endoplasmic reticulum stress response. Endoplasmic reticulum stress activated the expression of the ATF4 protein, upregulated the level of DDIT4, inhibited the MTOR signaling pathway, and caused cellular autophagy.

DDIT4 gene encodes a protein whose main action is to inhibit mTOR under stress conditions whilst several in vitro studies indicate that its expression favors cancer progression. We have previously described that DDIT4 expression is an independent prognostic factor for tripe negative breast cancer resistant to neoadjuvant chemotherapy. We herein report that high DDIT4 expression is related to the outcome (recurrence-free survival, time to progression and overall survival) in several cancer types. We performed in silico analysis in online platforms, in pooled datasets from KM Plotter and meta-analysis of individual datasets from SurvExpress. High levels of DDIT4 were significantly associated with a worse prognosis in acute myeloid leukemia, breast cancer, glioblastoma multiforme, colon, skin and lung cancer. Conversely, a high DDIT4 expression was associated with an improved prognostic in gastric cancer. DDIT4 was not associated with the outcome of ovarian cancers. Analysis with data from the Cell Miner Tool in 60 cancer cell lines indicated that although rapamycin activity was correlated with levels of MTOR, it is not influenced by DDIT4 expression. In summary, DDIT4 might serve as a novel prognostic biomarker in several malignancies. DDIT4 activity could be responsible for resistance to mTOR inhibitors and is a potential candidate for the development of targeted therapy.

This study was performed to investigate the global expression profile of microRNAs in distinct subpopulations of a human malignant mesothelioma cell line. Total RNAs were isolated from the sorted side population and non-side population of MS1. The RNAs were subjected to analysis using Affymetrix GeneChip microRNA Arrays. After data extraction and normalization, a subset of microRNAs defining cell subpopulations was identified using bioinformatics softwares. Based on the criteria of 2-fold difference and the p-value of < 0.05, a total of 95 microRNAs were differentially expressed in the side population compared to the non-side population. Functional ontology revealed that target genes of the miRNAs were categorized into various gene ontology terms, such as stem cell maintenance, cell proliferation, programmed cell death, cell migration, and cellular response to stress. The Kyoto Encyclopedia of Genes and Genomes analysis showed that ErbB-2 receptor tyrosine kinases signaling pathway was the most represented. Integrated analysis of MiRTarBase and RNA-seq identified 12 target genes of microRNAs defining side population, including DDIT4 and ROCK2. The present study indicates that a distinct set of microRNAs may be critically involved in the generation and maintenance of heterogeneous subpopulations of cancer cells. They could be a plausible target for the eradication of more aggressive cancer cell subpopulations.

The promyelocytic leukemia (PML) protein is an essential component of PML nuclear bodies (PML NBs) frequently lost in cancer. PML NBs coordinate chromosomal regions via modification of nuclear proteins that in turn may regulate genes in the vicinity of these bodies. However, few PML NB-associated genes have been identified. PML and PML NBs can also regulate mTOR and cell fate decisions in response to cellular stresses. We now demonstrate that PML depletion in U2OS cells or TERT-immortalized normal human diploid fibroblasts results in decreased expression of the mTOR inhibitor DDIT4 (REDD1). DNA and RNA immuno-FISH reveal that PML NBs are closely associated with actively transcribed DDIT4 loci, implicating these bodies in regulation of basal DDIT4 expression. Although PML silencing did reduce the sensitivity of U2OS cells to metabolic stress induced by metformin, PML loss did not inhibit the upregulation of DDIT4 in response to metformin, hypoxia-like (CoCl

Oncogenic human papillomaviruses (HPVs) are closely linked to major human malignancies, including cervical and head and neck cancers. It is widely assumed that HPV-positive cancer cells are under selection pressure to continuously express the viral E6/E7 oncogenes, that their intracellular p53 levels are reconstituted on E6/E7 repression, and that E6/E7 inhibition phenotypically results in cellular senescence. Here we show that hypoxic conditions, as are often found in subregions of cervical and head and neck cancers, enable HPV-positive cancer cells to escape from these regulatory principles: E6/E7 is efficiently repressed, yet, p53 levels do not increase. Moreover, E6/E7 repression under hypoxia does not result in cellular senescence, owing to hypoxia-associated impaired mechanistic target of rapamycin (mTOR) signaling via the inhibitory REDD1/TSC2 axis. Instead, a reversible growth arrest is induced that can be overcome by reoxygenation. Impairment of mTOR signaling also interfered with the senescence response of hypoxic HPV-positive cancer cells toward prosenescent chemotherapy in vitro. Collectively, these findings indicate that hypoxic HPV-positive cancer cells can induce a reversible state of dormancy, with decreased viral antigen synthesis and increased therapeutic resistance, and may serve as reservoirs for tumor recurrence on reoxygenation.

Receptor-PI3K-mTORC1 signaling and fatty acid synthase (FASN)-regulated lipid biosynthesis harbor numerous drug targets and are molecularly connected. We hypothesize that unraveling the mechanisms of pathway cross-talk will be useful for designing novel co-targeting strategies for ovarian cancer (OC). The impact of receptor-PI3K-mTORC1 onto FASN is already well-characterized. However, reverse actions-from FASN towards receptor-PI3K-mTORC1-are still elusive. We show that FASN-blockade impairs receptor-PI3K-mTORC1 signaling at multiple levels. Thin-layer chromatography and MALDI-MS/MS reveals that FASN-inhibitors (C75, G28UCM) augment polyunsaturated fatty acids and diminish signaling lipids diacylglycerol (DAG) and phosphatidylinositol 3,4,5-trisphosphate (PIP3) in OC cells (SKOV3, OVCAR-3, A2780, HOC-7). Western blotting and micropatterning demonstrate that FASN-blockers impair phosphorylation/expression of EGF-receptor/ERBB/HER and decrease GRB2-EGF-receptor recruitment leading to PI3K-AKT suppression. FASN-inhibitors activate stress response-genes HIF-1α-REDD1 (RTP801/DIG2/DDIT4) and AMPKα causing mTORC1- and S6-repression. We conclude that FASN-inhibitor-mediated blockade of receptor-PI3K-mTORC1 occurs due to a number of distinct but cooperating processes. Moreover, decrease of PI3K-mTORC1 abolishes cross-repression of MEK-ERK causing ERK activation. Consequently, the MEK-inhibitor selumetinib/AZD6244, in contrast to the PI3K/mTOR-inhibitor dactolisib/NVP-BEZ235, increases growth inhibition when given together with a FASN-blocker. We are the first to provide deep insight on how FASN-inhibition blocks ERBB-PI3K-mTORC1 activity at multiple molecular levels. Moreover, our data encourage therapeutic approaches using FASN-antagonists together with MEK-ERK-inhibitors.

Long non-coding RNA (lncRNA) plays an important role in tumorigenesis. However, the expression pattern and function of lncRNAs in laryngeal squamous cell carcinoma (LSCC) are still unclear. To investigate the aberrantly expressed lncRNAs and mRNAs in advanced LSCC, we screened lncRNA and mRNA expression profiles in 9 pairs of primary Stage IVA LSCC tissues and adjacent non-neoplastic tissues by lncRNA and mRNA integrated microarrays. Gene Ontology and pathway analysis were performed to find out the significant function and pathway of the differentially expressed mRNAs, gene-gene functional interaction network and ceRNA network were constructed to select core mRNAs, and lncRNA-mRNA expression correlation network was built to identify the interactions between lncRNA and mRNA. qRT-PCR was performed to further validate the expressions of selected lncRNAs and mRNAs in advanced LSCC. We found 1459 differentially expressed lncRNAs and 2381 differentially expressed mRNAs, including 846 up-regulated lncRNAs and 613 down-regulated lncRNAs, 1542 up-regulated mRNAs and 839 down-regulated mRNAs. The mRNAs ITGB1, HIF1A, and DDIT4 were selected as core mRNAs, which are mainly involved in biological processes, such as matrix organization, cell cycle, adhesion, and metabolic pathway. LncRNA-mRNA expression correlation network showed LncRNA NR_027340, MIR31HG were positively correlated with ITGB1, HIF1A respectively. LncRNA SOX2-OT was negatively correlated with DDIT4. qRT-PCR further validated the expression of these lncRNAs and mRNAs. The work provides convincing evidence that the identified lncRNAs and mRNAs are potential biomarkers in advanced LSCC for further future studies.

The hypoxic microenvironment is an important contributor of glioblastoma (GBM) aggressiveness via HIF1α, while tumour inflammation is profoundly influenced by FAT Atypical Cadherin (FAT1). This study was designed to explore the functional interaction and significance of FAT1 and HIF1α under severe hypoxia-mimicking tumour microenvironment in primary human tumours. We first identified a positive correlation of FAT1 with HIF1α and its target genes in GBM tumour specimens, revealing the significance of the FAT1-HIF1α axis in glioma cells. We found that severe hypoxia leads to an increased expression of FAT1 and HIF1α in U87MG and U373MG cells. To reveal the relevance of FAT1 under hypoxic conditions, we depleted endogenous FAT1 under hypoxia and found a substantial reduction in the expression of HIF1α and its downstream target genes like CA9, GLUT1, VEGFA, MCT4, HK2, BNIP3 and REDD1, as well as a significant reduction in the invasiveness in GBM cells. At the molecular level, under hypoxia the FAT1 depletion-associated reduction in HIF1α was due to compromised EGFR-Akt signaling as well as increased VHL-dependent proteasomal degradation of HIF1α. In brief, for the first time, these results reveal an upstream master regulatory role of FAT1 in the expression and role of HIF1α under hypoxic conditions and that FAT1-HIF1α axis controls the invasiveness of GBM. Hence, FAT1 represents a novel potential therapeutic target for GBM.

Ezrin is a member of the ERM (ezrin/radixin/moesin) family of proteins that links cortical cytoskeleton to the plasma membrane. High expression of ezrin correlates with poor prognosis and metastasis in osteosarcoma. In this study, to uncover specific cellular responses evoked by ezrin inhibition that can be used as a specific pharmacodynamic marker(s), we profiled global gene expression in osteosarcoma cells after treatment with small molecule ezrin inhibitors, NSC305787 and NSC668394. We identified and validated several up-regulated integrated stress response genes including PTGS2, ATF3, DDIT3, DDIT4, TRIB3, and ATF4 as novel ezrin-regulated transcripts. Analysis of transcriptional response in skin and peripheral blood mononuclear cells from NSC305787-treated mice compared with a control group revealed that, among those genes, the stress gene DDIT4/REDD1 may be used as a surrogate pharmacodynamic marker of ezrin inhibitor compound activity. In addition, we validated the anti-metastatic effects of NSC305787 in reducing the incidence of lung metastasis in a genetically engineered mouse model of osteosarcoma and evaluated the pharmacokinetics of NSC305787 and NSC668394 in mice. In conclusion, our findings suggest that cytoplasmic ezrin, previously considered a dormant and inactive protein, has important functions in regulating gene expression that may result in down-regulation of stress response genes.

The purpose of this study was to ascertain the mechanisms by which advanced prostate cancer cells resist bortezomib therapy. Several independent studies have shown that cells are protected from proteasome inhibition by increased autophagic activity. We investigated whether C/EBPβ, a transcription factor involved in the control of autophagic gene expression, regulates resistance to proteasome inhibition. In PC3 cells over-expressing C/EBPβ, turnover of autophagic substrates and expression of core autophagy genes were increased. Conversely, C/EBPβ knockdown suppressed autophagosome-lysosome fusion. We also found that C/EBPβ knockdown suppressed REDD1 expression to delay early autophagy, an effect rescued by exogenous REDD1. Cells with suppressed C/EBPβ levels showed delayed autophagy activation upon bortezomib treatment. Knockdown of C/EBPβ sensitized PC3 cells to bortezomib, and blockade of autophagy by chloroquine did not further increase cell death in cells expressing shRNA targeting C/EBPβ. Lastly, we observed a decreased growth of PC3 cells and xenografts with C/EBPβ knockdown and such xenografts were sensitized to bortezomib treatment. Our results demonstrate that C/EBPβ is a critical effector of autophagy via regulation of autolysosome formation and promotes resistance to proteasome inhibitor treatment by increasing autophagy.

Melatonin is implicated in various physiological functions, including anticancer activity. However, the mechanism(s) of its anticancer activity is not well understood. In the present study, we investigated the combined effects of melatonin and arsenic trioxide (ATO) on cell death in human breast cancer cells. Melatonin enhanced the ATO-induced apoptotic cell death via changes in the protein levels of Survivin, Bcl-2, and Bax, thus affecting cytochrome c release from the mitochondria to the cytosol. Interestingly, we found that the cell death induced by co-treatment with melatonin and ATO was mediated by sustained upregulation of Redd1, which was associated with increased production of reactive oxygen species (ROS). Combined treatment with melatonin and ATO induced the phosphorylation of JNK and p38 MAP kinase downstream from Redd1 expression. Rapamycin and S6K1 siRNA enhanced, while activation of mTORC1 by transfection with TSC2 siRNA suppressed the cell death induced by melatonin and ATO treatment. Taken together, our findings suggest that melatonin enhances ATO-induced apoptotic cell death via sustained upregulation of Redd1 expression and inhibition of mTORC1 upstream of the activation of the p38/JNK pathways in human breast cancer cells.

Lung cancer is the most common cause of cancer mortality and new cases are on the increase worldwide. However, the treatment of lung cancer remains unsatisfactory. Curcumin has been shown to induce cell death in many human cancer cells, including human lung cancer cells. However, the effects of curcumin on genetic mechanisms associated with these actions remain unclear. Curcumin (2 µM) was added to NCI-H460 human lung cancer cells and the cells were incubated for 24 h. Total RNA was extracted from isolated cells for cDNA synthesis, labeling, microarray hybridization and flour‑labeled cDNA hybridized on chip. Localized concentrations of fluorescent molecules were detected and quantified using Expression Console software (Affymetrix) with default RMA parameters. GeneGo software was used for the key genes involved and their possible interaction pathways. The results showed that ~170 genes were significantly upregulated and 577 genes were significantly downregulated in curcumin‑treated cells. Specifically, the up‑ and downregulated genes included CCNE2, associated with DNA damage; ID3, associated with cell survival and 146 genes with a >2- to 3-fold change including the TP53INP1 gene, associated with DNA damage; CDC6, CDCA5, TAKMIP2, CDK14, CDK5, CDCA76, CDC25A, CDC5L and SKP2, associated with cell cycle; the CARD6, ID1 and ID2 genes, associated with cell survival and the BRMS1L, associated with cell migration and invasion. Additionally, 59 downregulated genes exhibited a >4-fold change, including the DDIT3 gene, associated with DNA damage; while 97 genes had a >3- to 4-fold change including the DDIT4 gene, associated with DNA damage; the CCPG1 gene, associated with cell cycle and 321 genes with a >2- to 3-fold including the GADD45A and CGREF1 genes, associated with DNA damage; the CCPG1 gene, associated with cell cycle, the TNFRSF10B, GAS5, TSSC1 and TNFRSF11B gene, associated with cell survival and the ARHAP29 and CADM2 genes, associated with cell migration and invasion. In conclusion, gene alterations provide information regarding the cytotoxic mechanism of curcumin at the genetic level and provide additional biomarkers or targets for the treatment of human lung cancer.

In the past decade, no significant improvement has been made in chemotherapy for osteosarcoma (OS). To develop improved agents against OS, we screened 70 species of medicinal plants and treated two human OS cell lines with different agent concentrations. We then examined cell viability using the MTT assay. Results showed that a candidate plant, particularly the rhizomes of Anemone altaica Fisch. ex C. A. Mey aqueous extract (AAE), suppressed the viability of HOS and U2OS cells in a concentration-dependent manner. Flow cytometry analysis revealed that AAE significantly increased the amount of cell shrinkage (Sub-G1 fragments) in HOS and U2OS cells. Moreover, AAE increased cytosolic cytochrome c and Bax, but decreased Bcl-2. The amount of cleaved caspase-3 and poly-(ADP-ribose) polymerase-1 (PARP-1) were significantly increased. AAE suppressed the growth of HOS and U2OS through the intrinsic apoptotic pathway. Data suggest that AAE is cytotoxic to HOS and U2OS cells and has no significant influence on human osteoblast hFOB cells. The high mRNA levels of apoptosis-related factors (PPP1R15A, SQSTM1, HSPA1B, and DDIT4) and cellular proliferation markers (SKA2 and BUB1B) were significantly altered by the AAE treatment of HOS and U2OS cells. Results show that the anticancer activity of AAE could up-regulate the expression of a cluster of genes, especially those in the apoptosis-related factor family and caspase family. Thus, AAE has great potential as a useful therapeutic drug for human OS.

YAP (yes-associated protein) and TAZ are oncogenic transcriptional co-activators downstream of the Hippo tumor-suppressor pathway. However, whether YAP and/or TAZ (YAP/TAZ) engage in transcriptional co-repression remains relatively unexplored. Here, we directly demonstrated that YAP/TAZ represses numerous target genes, including tumor-suppressor genes such as DDIT4 (DNA-damage-inducible transcript 4) and Trail (TNF-related apoptosis-inducing ligand). Mechanistically, the repressor function of YAP/TAZ requires TEAD (TEA domain) transcription factors. A YAP/TAZ-TEAD complex recruits the NuRD complex to deacetylate histones and alters nucleosome occupancy at target genes. Functionally, repression of DDIT4 and Trail by YAP/TAZ is required for mTORC1 (mechanistic target of rapamycin complex 1) activation and cell survival, respectively. Our demonstration of the transcriptional co-repressor activity of YAP/TAZ opens a new avenue for understanding the Hippo signaling pathway.

Cantharidin (CTD) induces cytotoxic effects in different types of human cancer cell; however, to date, there have been no studies on the effects of CTD on gene expression in human lung cancer cells and the potential associated signaling pathways. Therefore, the present study aimed to investigate how CTD affects the expression of key genes and functional pathways of human H460 lung cancer cells using complementary DNA microarray analysis. Human H460 lung cancer cells were cultured for 24 h in the presence or absence of 10 µM CTD; gene expression was then examined using microarray analysis. The results indicated that 8 genes were upregulated > 4-fold, 29 genes were upregulated >3-4-fold and 156 genes were upregulated >2-3-fold. In addition, 1 gene was downregulated >4 fold, 14 genes were downregulated >3-4-fold and 150 genes were downregulated >2-3 fold in H460 cells following exposure to CTD. It was found that CTD affected DNA damage genes, including DNIT3 and GADD45A, which were upregulated 2.26- and 2.60-fold, respectively, as well as DdiT4, which was downregulated 3.14-fold. In addition, the expression of genes associated with the cell cycle progression were altered, including CCND2, CDKL3 and RASA4, which were upregulated 2.72-, 2.19- and 2.72-fold, respectively; however, CDC42EP3 was downregulated 2.16-fold. Furthermore, apoptosis-associated genes were differentially expressed, including CARD6, which was upregulated 3.54-fold. In conclusion, the present study demonstrated that CTD affected the expression of genes associated with DNA damage, cell cycle progression and apoptotic cell death in human lung cancer H460 cells.

We previously reported that a pan-PAD inhibitor, YW3-56, activates p53 target genes to inhibit cancer growth. However, the p53-independent anticancer activity and molecular mechanisms of YW3-56 remain largely elusive. Here, gene expression analyses found that ATF4 target genes involved in endoplasmic reticulum (ER) stress response were activated by YW3-56. Depletion of ATF4 greatly attenuated YW3-56-mediated activation of the mTORC1 regulatory genes SESN2 and DDIT4. Using the ChIP-exo method, high-resolution genomic binding sites of ATF4 and CEBPB responsive to YW3-56 treatment were generated. In human breast cancer cells, YW3-56-mediated cell death features mitochondria depletion and autophagy perturbation. Moreover, YW3-56 treatment effectively inhibits the growth of triple-negative breast cancer xenograft tumors in nude mice. Taken together, we unveiled the anticancer mechanisms and therapeutic potentials of the pan-PAD inhibitor YW3-56.

Baicalein is a natural flavone that exhibits anticancer properties. Using microarrays we found that DDIT4 was the highest transcript induced by baicalein in cancer cells. We confirmed in multiple cancer cell lines large, dose-related expression of DDIT4 by quantitative RT-PCR and immunoblot, which correlates with growth inhibition. Time course experiments demonstrate that DDIT4 is rapidly inducible, with high expression maintained for several days in vitro. Induction of DDIT4 expression is p53 independent based on evaluation of p53 knockout cells. Since DDIT4 is known to inhibit mTORC1 activity we confirmed that baicalein suppresses phosphorylation of mTORC1 targets. Using RNA interference we demonstrate that mTORC1 activity and growth inhibition by baicalein is attenuated by knockdown of DDIT4. We furthermore demonstrate suppression of established tumors by baicalein in a mouse model of breast cancer with increased DDIT4 expression in the tumors. Finally, we demonstrate that baicalein upregulates DDIT4 and causes mTORC1 and growth inhibition in platinum resistant cancer cells in marked contrast to platinum chemotherapy treatment. These studies demonstrate that baicalein inhibits mTORC1 through DDIT4 expression, and may be useful in cancer chemotherapy and chemoprevention.

MicroRNAome analyses have shown microRNA-630 (miR-630) to be involved in the regulation of apoptosis. However, its apoptotic role is still debated and its participation in DNA replication is unknown. Here, we demonstrate that miR-630 inhibits cell proliferation by targeting cell-cycle kinase 7 (CDC7) kinase, but maintains the apoptotic balance by targeting multiple activators of apoptosis under genotoxic stress. We identified a novel regulatory mechanism of CDC7 gene expression, in which miR-630 downregulated CDC7 expression by recognizing and binding to four binding sites in CDC7 3'-UTR. We found that miR-630 was highly expressed in A549 and NIH3T3 cells where CDC7 was downregulated, but lower in H1299, MCF7, MDA-MB-231, HeLa and 2BS cells where CDC7 was upregulated. Furthermore, the induction of miR-630 occurred commonly in a variety of human cancer and immortalized cells in response to genotoxic agents. Importantly, downregulation of CDC7 by miR-630 was associated with cisplatin (CIS)-induced inhibitory proliferation in A549 cells. Mechanistically, miR-630 exerted its inhibitory proliferation by blocking CDC7-mediated initiation of DNA synthesis and by inducing G1 arrest, but maintains apoptotic balance under CIS exposure. On the one hand, miR-630 promoted apoptosis by downregulation of CDC7; on the other hand, it reduced apoptosis by downregulating several apoptotic modulators such as PARP3, DDIT4, EP300 and EP300 downstream effector p53, thereby maintaining the apoptotic balance. Our data indicate that miR-630 has a bimodal role in the regulation of apoptosis in response to DNA damage. Our data also support the notion that a certain mRNA can be targeted by several miRNAs, and in particular an miRNA may target a set of mRNAs. These data afford a comprehensive view of microRNA-dependent control of gene expression in the regulation of apoptosis under genotoxic stress.

Targeted degradation of proteins through the ubiquitin-proteasome system (UPS) via the activities of E3 ubiquitin ligases regulates diverse cellular processes, and misregulation of these enzymes contributes to the pathogenesis of human diseases. One of the challenges facing the UPS field is to delineate the complete cohort of substrates for a particular E3 ligase. Advances in mass spectrometry and the development of antibodies recognizing the Lys-ϵ-Gly-Gly (diGly) remnant from ubiquitinated proteins following trypsinolysis have provided a tool to address this question. We implemented an inducible loss of function approach in combination with quantitative diGly proteomics to find novel substrates of HUWE1 (HECT, UBA, and WWE domain containing 1, E3 ubiquitin protein ligase), an E3 ligase implicated in cancer and intellectual disabilities. diGly proteomics results led to the identification of DNA damage-inducible transcript 4 (DDIT4) as a putative HUWE1 substrate. Cell-based assays demonstrated that HUWE1 interacts with and regulates ubiquitination and stability of DDIT4. Together these data suggest a model in which HUWE1 mediates DDIT4 proteasomal degradation. Our results demonstrate proof of concept that inducible knockdown of an E3 ligase in combination with diGly proteomics provides a potentially advantageous method for identifying novel E3 substrates that may help to identify candidates for therapeutic modulation in the UPS.

Osteosarcoma (OS) is a type of bone cancer. Eighty percent of this tumor will metastasize to the lungs or liver, and as a result, patients generally need chemotherapy to improve survival possibility. Recently, antitumor activity has been reported in Ocimum gratissimum aqueous extract (OGE), which has been the focus of recent extensive studies on therapeutic strategies due to its antioxidant properties. We performed pharmacogenomics analyses for the effect of OGE on human osteosarcoma U2-OS and HOS cell growth. Cell viability, Western blot and flow cytometry analysis were performed before performing pharmacogenomics analyses for the effect of OGE on human osteosarcoma U2-OS and HOS cell growth, including cDNA microarray and RT-PCR assays. Cell viability assays revealed that OGE significantly and dose-dependently decreased the viability of U2-OS and HOS cells. Increases in cell shrinkage, Sub-G1 fragments and the activation of caspase 3 indicated that OGE induced cell apoptosis in U2-OS and HOS cells. There was no change in human osteoblast hFOS cells. cDNA microarray assay demonstrated that the expression of cell cycle regulators, apoptosis-related factors and cell proliferation markers were all modified by OGE treatment. RT-PCR analysis also confirmed the down-regulation of SKA2 and BUB1B, and the up-regulation of PPP1R15A, SQSTM1, HSPA1B, and DDIT4 by OGE treatment. The finding of anticancer activity in OGE and the identification of some potential target genes raise the expectation that OGE may become a useful therapeutic drug for human OS.

Lung cancer is the leading cause of cancer related death and there is no effective treatment to date. Bufalin has been shown effective in inducing apoptosis and DNA damage in lung cancer cells. However, the genetic mechanisms underlying these actions have not been elucidated yet. Cultured NCI-H460 cells were treated with or without 2 μM of bufalin for 24 h. The total RNA was extracted from each treatment for cDNA synthesis and labeling, microarray hybridization, and then followed by flour-labeled cDNA hybridized on chip. The localized concentrations of fluorescent molecules were detected and quantitated and analyzed by Expression Console software (Affymetrix) with default RMA parameters. The key genes involved and their possible interaction pathways were mapped by GeneGo software. About 165 apoptosis-related genes were affected. CASP9 was up-regulated by 5.51 fold and THAP1 by 2.75-fold while CCAR1 was down-regulated by 2.24 fold. 107 genes related to DNA damage/repair were affected. MDC1 was down-regulated by 2.22-fold, DDIT4 by 2.52 fold while GADD45B up-regulated by 3.72 fold. 201 genes related to cell cycles were affected. CCPG1 was down-regulated by 2.11 fold and CDCA7L by 2.71 fold. Many genes about apoptosis, cell cycle regulation and DNA repair are changed significantly following bufalin treatment in NCI-H460 cells. These changes provide an in depth understanding of cytotoxic mechanism of bufalin in genetic level and also offer many potentially useful biomarkers for diagnosis and treatment of lung cancer in future.

Recent advances in non-protein coding part of human genome analysis have discovered extensive transcription of large RNA transcripts that lack of coding protein function, termed long noncoding RNAs (lncRNAs). It is becoming evident that lncRNAs may be an important class of pervasive genes involved in carcinogenesis and metastasis. However, the biological and molecular mechanisms of lncRNAs in diverse diseases are not yet fully understood. Thus, it is anticipated that more efforts should be made to clarify the lncRNAs world. Moreover, accumulating studies have demonstrated that a class of lncRNAs are dysregulated in hepatocellular carcinoma(HCC) and closely related with tumorigenesis, metastasis, prognosis or diagnosis. In this review, we will briefly discuss the regulation and functional role of lncRNAs in HCC, therefore evaluating the potential of lncRNAs as prospective novel therapeutic targets in HCC.

Energy metabolism plasticity enables stemness programs during the reprogramming of somatic cells to an induced pluripotent stem cell (iPSC) state. This relationship may introduce a new era in the understanding of Warburg's theory on the metabolic origin of cancer at the level of cancer stem cells (CSCs). Here, we used Yamanaka's stem cell technology in an attempt to create stable CSC research lines in which to dissect the transcriptional control of mTOR--the master switch of cellular catabolism and anabolism--in CSC-like states. The rare colonies with iPSC-like morphology, obtained following the viral transduction of the Oct4, Sox2, Klf4, and c-Myc (OSKM) stemness factors into MCF-7 luminal-like breast cancer cells (MCF-7/Rep), demonstrated an intermediate state between cancer cells and bona fide iPSCs. MCF-7/Rep cells notably overexpressed SOX2 and stage-specific embryonic antigen (SSEA)-4 proteins; however, other stemness-related markers (OCT4, NANOG, SSEA-1, TRA-1-60, and TRA-1-81) were found at low to moderate levels. The transcriptional analyses of OSKM factors confirmed the strong but unique reactivation of the endogenous Sox2 stemness gene accompanied by the silencing of the exogenous Sox2 transgene in MCF-7/Rep cells. Some but not all MCF-7/Rep cells acquired strong alkaline phosphatase (AP) activity compared with MCF-7 parental cells. SOX2-overexpressing MCF-7/Rep cells contained drastically higher percentages of CD44(+) and ALDEFLUOR-stained ALDH(bright) cells than MCF-7 parental cells. The overlap between differentially expressed mTOR signaling-related genes in 3 different SOX2-overexpressing CSC-like cell lines revealed a notable downregulation of 3 genes, PRKAA1 (which codes for the catalytic α 1 subunit of AMPK), DDIT4/REDD1 (a stress response gene that operates as a negative regulator of mTOR), and DEPTOR (a naturally occurring endogenous inhibitor of mTOR activity). The insulin-receptor gene (INSR) was differentially upregulated in MCF-7/Rep cells. Consistent with the downregulation of AMPK expression, immunoblotting procedures confirmed upregulation of p70S6K and increased phosphorylation of mTOR in Sox2-overexpressing CSC-like cell populations. Using an in vitro model of the de novo generation of CSC-like states through the nuclear reprogramming of an established breast cancer cell line, we reveal that the transcriptional suppression of mTOR repressors is an intrinsic process occurring during the acquisition of CSC-like properties by differentiated populations of luminal-like breast cancer cells. This approach may provide a new path for obtaining information about preventing the appearance of CSCs through the modulation of the AMPK/mTOR pathway.

Angiogenesis is essential for tumor development and progression. Genetic variation in angiogenesis-related genes may influence breast carcinogenesis. We evaluated dietary factors associated with oxidative balance, DDIT4 (one SNP), FLT1 (35 SNPs), HIF1A (four SNPs), KDR (19 SNPs), MPO (one SNP), NOS2A (15 SNPs), TEK (40 SNPs) and VEGFA (eight SNPs) and breast cancer risk among Hispanic (2,111 cases and 2,597 controls) and non-Hispanic white (1,481 cases and 1,586 controls) women in the Breast Cancer Health Disparities Study. Adaptive rank truncated product (ARTP) analysis was used to determine gene and pathway significance with breast cancer. TEK was associated with breast cancer overall (pARTP = 0.03) and with breast cancer survival (pARTP = 0.01). KDR was of borderline significance overall (pARTP = 0.07), although significantly associated with breast cancer in both low and intermediate Native American (NA) ancestry groups (pARTP = 0.02) and estrogen receptor (ER)+/progesterone receptor (PR)- tumor phenotype (pARTP = 0.008). Both VEGFA and NOS2A were associated with ER-/PR- tumor phenotype (pARTP = 0.01 and pARTP = 0.04, respectively). FLT1 was associated with breast cancer survival among those with low NA ancestry (pARTP = 0.009). With respect to diet, having a higher dietary oxidative balance score (DOBS) was significantly associated with lower breast cancer risk [odds ratio (OR) 0.74, 95% confidence interval (CI) 0.64-0.84], with the strongest associations observed for women with the highest NA ancestry (OR 0.44, 95% CI 0.30-0.65). We observed few interactions between DOBS and angiogenesis-related genes. Our data suggest that dietary factors and genetic variation in angiogenesis-related genes contribute to breast cancer carcinogenesis.