Christos Polytarchou

Lin28A and Lin28B Inhibit let-7 MicroRNA Biogenesis by Distinct Mechanisms

Lin28A and Lin28B selectively block the expression of let-7 microRNAs and function as oncogenes in a variety of human cancers. Lin28A recruits a TUTase (Zcchc11/TUT4) to let-7 precursors to block processing by Dicer in the cell cytoplasm. Here we find that unlike Lin28A, Lin28B represses let-7 processing through a Zcchc11-independent mechanism. Lin28B functions in the nucleus by sequestering primary let-7 transcripts and inhibiting their processing by the Microprocessor. The inhibitory effects of Zcchc11 depletion on the tumorigenic capacity and metastatic potential of human cancer cells and xenografts are restricted to Lin28A-expressing tumors. Furthermore, the majority of human colon and breast tumors analyzed exclusively express either Lin28A or Lin28B. Lin28A is expressed in HER2-overexpressing breast tumors, whereas Lin28B expression characterizes triple-negative breast tumors. Overall our results illuminate the distinct mechanisms by which Lin28A and Lin28B function and have implications for the development of new strategies for cancer therapy.

An HNF4α-miRNA Inflammatory Feedback Circuit Regulates Hepatocellular Oncogenesis

Hepatocyte nuclear factor 4α (HNF4α) is essential for liver development and hepatocyte function. Here, we show that transient inhibition of HNF4α initiates hepatocellular transformation through a microRNA-inflammatory feedback loop circuit consisting of miR-124, IL6R, STAT3, miR-24, and miR-629. Moreover, we show that, once this circuit is activated, it maintains suppression of HNF4α and sustains oncogenesis. Systemic administration of miR-124, which modulates inflammatory signaling, prevents and suppresses hepatocellular carcinogenesis by inducing tumor-specific apoptosis without toxic side effects. As we also show that this HNF4α circuit is perturbed in human hepatocellular carcinomas, our data raise the possibility that manipulation of this microRNA feedback-inflammatory loop has therapeutic potential for treating liver cancer.Hepatocyte nuclear factor 4α (HNF4α) is essential for liver development and hepatocyte function. Here, we show that transient inhibition of HNF4α initiates hepatocellular transformation through a microRNA-inflammatory feedback loop circuit consisting of miR-124, IL6R, STAT3, miR-24, and miR-629. Moreover, we show that, once this circuit is activated, it maintains suppression of HNF4α and sustains oncogenesis. Systemic administration of miR-124, which modulates inflammatory signaling, prevents and suppresses hepatocellular carcinogenesis by inducing tumor-specific apoptosis without toxic side effects. As we also show that this HNF4α circuit is perturbed in human hepatocellular carcinomas, our data raise the possibility that manipulation of this microRNA feedback-inflammatory loop has therapeutic potential for treating liver cancer.

Hydrogen Peroxide Stimulates Proliferation and Migration of Human Prostate Cancer Cells through Activation of Activator Protein-1 and Up-regulation of the Heparin Affin Regulatory Peptide Gene

It is becoming increasingly recognized that hydrogen peroxide (HP) plays a role in cell proliferation and migration. In the present study we found that exogenous HP significantly induced human prostate cancer LNCaP cell proliferation and migration. Heparin affin regulatory peptide (HARP) seems to be involved in the stimulatory effect of HP, because the latter had no effect on stably transfected LNCaP cells that did not express HARP. Moreover, HP significantly increased HARP mRNA and protein amounts in a concentration- and time-dependent manner. Curcumin and activator protein-1 (AP-1) decoy oligonucleotides abrogated both HP-induced HARP expression and LNCaP cell proliferation and migration. HP increased luciferase activity of the 5′-flanking region of the HARP gene introduced in a reporter gene vector, an effect that was abolished when even one of the two putative AP-1 binding sites of the HARP promoter was mutated. The effect of HP seems to be due to the binding of Fra-1, JunD, and phospho-c-Jun to the HARP promoter. These results support the notion that HARP is important for human prostate cancer cell proliferation and migration, establish the role of AP-1 in the up-regulation of HARP expression by low concentrations of HP, and characterize the AP-1 dimers involved.

An integrated transcriptional regulatory circuit that reinforces the breast cancer stem cell state

Cancer stem-like cells (CSCs) are a highly tumorigenic cell type present as a minority population in developmentally diverse tumors and cell lines. Using a genetic screen in an inducible model of CSC formation in a breast cell line, we identify microRNAs (miRNAs) that inhibit CSC growth and are down-regulated in CSCs. Aside from the previously identified miR-200 family, these include the miR-15/16 (miR-16, miR-15b) and miR-103/107 (miR-103, miR-107) families as well as miR-145, miR-335, and miR-128b. Interestingly, these miRNAs affect common target genes that encode the Bmi1 and Suz12 components of the polycomb repressor complexes as well as the DNA-binding transcription factors Zeb1, Zeb2, and Klf4. Conversely, expression of the CSC-modulating miRNAs is inhibited by Zeb1 and Zeb2. There is an inverse relationship between the levels of CSC-regulating miRNAs and their respective targets in samples from triple-negative breast cancer patients, providing evidence for the relevance of these interactions in human cancer. In addition, combinatorial overexpression of these miRNAs progressively attenuates the growth of CSCs derived from triple-negative breast cancers. These observations suggest that CSC formation and function are reinforced by an integrated regulatory circuit of miRNAs, transcription factors, and chromatin-modifying activities that can act as a bistable switch to drive cells into either the CSC or the nonstem state within the population of cancer cells.

Akt2 regulates all Akt isoforms and promotes resistance to hypoxia through induction of miR-21 upon oxygen deprivation

The growth and survival of tumor cells in an unfavorable hypoxic environment depend upon their adaptability. Here, we show that both normal and tumor cells expressing the protein kinase Akt2 are more resistant to hypoxia than cells expressing Akt1 or Akt3. This is due to the differential regulation of microRNA (miR) 21, which is upregulated by hypoxia only in Akt2-expressing cells. By upregulating miR-21 upon oxygen deprivation, Akt2 downregulates PTEN and activates all three Akt isoforms. miR-21 also targets PDCD4 and Sprouty 1 (Spry1), and the combined downregulation of these proteins with PTEN is sufficient to confer resistance to hypoxia. Furthermore, the miR-21 induction by Akt2 during hypoxia depends upon the binding of NF-κB, cAMP responsive element-binding protein (CREB), and CBP/p300 to the miR-21 promoter, in addition to the regional acetylation of histone H3K9, all of which are under the control of Akt2. Analysis of the Akt2/miR-21 pathway in hypoxic MMTV-PyMT-induced mouse mammary adenocarcinomas and human ovarian carcinomas confirmed the activity of the pathway in vivo. Taken together, this study identifies a novel Akt2-dependent pathway that is activated by hypoxia and promotes tumor resistance via induction of miR-21.

Antioxidants inhibit angiogenesis in vivo through down-regulation of nitric oxide synthase expression and activity.

Although reactive oxygen species (ROS) participate in many cellular mechanisms, only few data exist concerning their involvement in physiological angiogenesis. The aim of the present work was to elucidate possible mechanisms through which ROS affect angiogenesis in vivo, using the model of the chicken embryo chorioallantoic membrane (CAM). Superoxide dismutase (SOD) and its membrane permeable mimetic tempol, dose dependently decreased angiogenesis and down-regulated inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) production. The NADPH oxidase inhibitors, 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF) and apocynin, but not allopurinol, also had a dose dependent inhibitory effect on angiogenesis and NO production in vivo. Catalase and the intracellular hydrogen peroxide (H2O2) scavenger sodium pyruvate decreased, while H2O2 increased in a dose-dependent manner the number of CAM blood vessels, as well as the expression and activity of iNOS. Dexamethasone, which down-regulated NO production by iNOS and l-NAME, but not d-NAME, dose dependently decreased angiogenesis in vivo. These data suggest that antioxidants affect physiological angiogenesis in vivo, through regulation of NOS expression and activity.

The protein kinase Akt1 regulates the interferon response through phosphorylation of the transcriptional repressor EMSY

The protein kinases Akt1, Akt2, and Akt3 possess nonredundant signaling properties, few of which have been investigated. Here, we present evidence for an Akt1-dependent pathway that controls interferon (IFN)-regulated gene expression and antiviral immunity. The target of this pathway is EMSY, an oncogenic interacting partner of BRCA2 that functions as a transcriptional repressor. Overexpression of EMSY in hTERT-immortalized mammary epithelial cells, and in breast and ovarian carcinoma cell lines, represses IFN-stimulated genes (ISGs) in a BRCA2-dependent manner, whereas its knockdown has the opposite effect. EMSY binds to the promoters of ISGs, suggesting that EMSY functions as a direct transcriptional repressor. Akt1, but not Akt2, phosphorylates EMSY at Ser209, relieving EMSY-mediated ISG repression. The Akt1/EMSY/ISG pathway is activated by both viral infection and IFN, and it inhibits the replication of HSV-1 and vesicular stomatitis virus (VSV). Collectively, these data define an Akt1-dependent pathway that contributes to the full activation of ISGs by relieving their repression by EMSY and BRCA2.

MicroRNA214 Is Associated With Progression of Ulcerative Colitis, and Inhibition Reduces Development of Colitis and Colitis-Associated Cancer in Mice

BACKGROUND & AIMS Persistent activation of the inflammatory response contributes to the development of inflammatory bowel diseases, which increase the risk of colorectal cancer. We aimed to identify microRNAs that regulate inflammation during the development of ulcerative colitis (UC) and progression to colitis-associated colon cancer (CAC). METHODS We performed a quantitative polymerase chain reaction analysis to measure microRNAs in 401 colon specimens from patients with UC, Crohns disease, irritable bowel syndrome, sporadic colorectal cancer, or CAC, as well as subjects without these disorders (controls); levels were correlated with clinical features and disease activity of patients. Colitis was induced in mice by administration of dextran sodium sulfate (DSS), and carcinogenesis was induced by addition of azoxymethane; some mice also were given an inhibitor of microRNA214 (miR214). RESULTS A high-throughput functional screen of the human microRNAome found that miR214 regulated the activity of nuclear factor-κB. Higher levels of miR214 were detected in colon tissues from patients with active UC or CAC than from patients with other disorders or controls and correlated with disease progression. Bioinformatic and genome-wide profile analyses showed that miR214 activates an inflammatory response and is amplified through a feedback loop circuit mediated by phosphatase and tensin homolog (PTEN) and PDZ and LIM domain 2 (PDLIM2). Interleukin-6 induced signal transducer and activator of transcription 3 (STAT3)-mediated transcription of miR214. A miR214 chemical inhibitor blocked this circuit and reduced the severity of DSS-induced colitis in mice, as well as the number and size of tumors that formed in mice given azoxymethane and DSS. In fresh colonic biopsy specimens from patients with active UC, the miR214 inhibitor reduced inflammation by increasing levels of PDLIM2 and PTEN. CONCLUSIONS Interleukin-6 up-regulates STAT3-mediated transcription of miR214 in colon tissues, which reduces levels of PDLIM2 and PTEN, increases phosphorylation of AKT, and activates nuclear factor-κB. The activity of this circuit correlates with disease activity in patients with UC and progression to colorectal cancer.

miRNAs link metabolic reprogramming to oncogenesis

The most profound biochemical phenotype of cancer cells is their ability to metabolize glucose to lactate, even under aerobic conditions. This alternative metabolic circuitry is sufficient to support the biosynthetic and energy requirements for cancer cell proliferation and metastasis. Alterations in oncogenes and tumor-suppressor genes are involved in the metabolic switch of cancer cells to aerobic glycolysis, increased glutaminolysis, and fatty acid biosynthesis. miRNAs mediate fine-tuning of genes involved directly or indirectly in cancer metabolism. In this review we discuss the regulatory role of miRNAs on enzymes, signaling pathways, and transcription factors involved in glucose and lipid metabolism. We further consider the therapeutic potential of metabolism-related miRNAs in cancer.

The JmjC Domain Histone Demethylase Ndy1 Regulates Redox Homeostasis and Protects Cells from Oxidative Stress

ABSTRACT The histone H3 demethylase Ndy1/KDM2B protects cells from replicative senescence. Changes in the metabolism of reactive oxygen species (ROS) are important for establishing senescence, suggesting that Ndy1 may play a role in redox regulation. Here we show that Ndy1 protects from H2O2-induced apoptosis and G2/M arrest and inhibits ROS-mediated signaling and DNA damage, while knockdown of Ndy1 has the opposite effects. Consistent with these observations, whereas Ndy1 overexpression promotes H2O2 detoxification, Ndy1 knockdown inhibits it. Ndy1 promotes the expression of genes encoding the antioxidant enzymes aminoadipic semialdehyde synthase (Aass), NAD(P)H quinone oxidoreductase-1 (Nqo1), peroxiredoxin-4 (Prdx4), and serine peptidase inhibitor b1b (Serpinb1b) and represses the expression of interleukin-19. At least two of these genes (Nqo1 and Prdx4) are regulated directly by Ndy1, which binds to specific sites within their promoters and demethylates promoter-associated histone H3 dimethylated at K36 and histone H3 trimethylated at K4. Simultaneous knockdown of Aass, Nqo1, Prdx4, and Serpinb1b in Ndy1-expressing cells to levels equivalent to those detected in control cells was sufficient to suppress the Ndy1 redox phenotype.