Nutrient sensor O-GlcNAc transferase controls cancer lipid metabolism via SREBP-1 regulation.

Elevated O-GlcNAcylation is associated with disease states such as diabetes and cancer. O-GlcNAc transferase (OGT) is elevated in multiple cancers and inhibition of this enzyme genetically or pharmacologically inhibits oncogenesis. Here we show that O-GlcNAcylation modulates lipid metabolism in cancer cells. OGT regulates expression of the master lipid regulator the transcription factor sterol regulatory element binding protein 1 (SREBP-1) and its transcriptional targets both in cancer and lipogenic tissue. OGT regulates SREBP-1 protein expression via AMP-activated protein kinase (AMPK). SREBP-1 is critical for OGT-mediated regulation of cell survival and of lipid synthesis, as overexpression of SREBP-1 rescues lipogenic defects associated with OGT suppression, and tumor growth in vitro and in vivo. These results unravel a previously unidentified link between O-GlcNAcylation, lipid metabolism and the regulation of SREBP-1 in cancer and suggests a crucial role for O-GlcNAc signaling in transducing nutritional state to regulate lipid metabolism.

O-GlcNAcylation regulates breast cancer metastasis via SIRT1 modulation of FOXM1 pathway.

Tumors utilize aerobic glycolysis to support growth and invasion. However, the molecular mechanisms that link metabolism with invasion are not well understood. The nutrient sensor O-linked-ß-N-acetylglucosamine (O-GlcNAc) transferase (OGT) modifies intracellular proteins with N-acetylglucosamine. Cancers display elevated O-GlcNAcylation and suppression of O-GlcNAcylation inhibits cancer invasion and metastasis. Here, we show that the regulation of cancer invasion by OGT is dependent on the NAD+-dependent deacetylase SIRT1. Reducing O-GlcNAcylation elevates SIRT1 levels and activity in an AMPK (AMP-activated protein kinase α)-dependent manner. Reduced O-GlcNAcylation in cancer cells leads to SIRT1-mediated proteasomal degradation of oncogenic transcription factor FOXM1 in an MEK/ERK-dependent manner. SIRT1 is critical for OGT-mediated regulation of FOXM1 ubiquitination and reducing SIRT1 activity reverses OGT-mediated regulation of FOXM1. Moreover, we show that SIRT1 levels are required for OGT-mediated regulation of invasion and metastasis in breast cancer cells. Thus, O-GlcNAcylation is a central component linking metabolism to invasion and metastasis via an SIRT1/ERK/FOXM1 axis.

ErbB2, FoxM1 and 14-3-3ζ prime breast cancer cells for invasion in response to ionizing radiation.

ErbB2 is frequently highly expressed in premalignant breast cancers, including ductal carcinoma in situ (DCIS); however, little is known about the signals or pathways it contributes to progression into the invasive/malignant state. Radiotherapy is often used to treat early premalignant lesions regardless of ErbB2 status. Here, we show that clinically relevant doses of ionizing radiation (IR)-induce cellular invasion of ErbB2-expressing breast cancer cells, as well as MCF10A cells overexpressing ErbB2. ErbB2-negative breast cancer cells, such as MCF7 and T47D, do not invade following treatment with IR nor do MCF10A cells overexpressing epidermal growth factor receptor. ErbB2 becomes phosphorylated at tyrosine 877 in a dose- and time- dependent manner following exposure to X-rays, and activates downstream signaling cascades including PI3K/Akt. Inhibition of these pathways, as well as inhibition of reactive oxygen species (ROS) with antioxidants, prevents IR-induced invasion. Activation of ErbB2-dependent signaling results in upregulation of the forkhead family transcription factor, FoxM1, and its transcriptional targets, including matrix metalloproteinase 2 (MMP2). Inhibition of FoxM1 by RNA interference prevented induction of invasion by IR, and overexpression of FoxM1 in MCF10A cells was sufficient to promote IR-induced invasion. Moreover, we found that 14-3-3ζ is also upregulated by IR in cancer cells in a ROS-dependent manner, is required for IR-induced invasion in ErbB2-positive breast cancer cells and together with FoxM1 is sufficient for invasion in ErbB2-negative breast cancer cells. Thus, our data show that IR-mediated activation of ErbB2 and induction of 14-3-3ζ collaborate to regulate FoxM1 and promote invasion of breast cancer cells and furthermore, may serve as therapeutic targets to enhance radiosensitivity of breast cancers.

HINCUTs in cancer: hypoxia-induced noncoding ultraconserved transcripts.

Recent data have linked hypoxia, a classic feature of the tumor microenvironment, to the function of specific microRNAs (miRNAs); however, whether hypoxia affects other types of noncoding transcripts is currently unknown. Starting from a genome-wide expression profiling, we demonstrate for the first time a functional link between oxygen deprivation and the modulation of long noncoding transcripts from ultraconserved regions, termed transcribed-ultraconserved regions (T-UCRs). Interestingly, several hypoxia-upregulated T-UCRs, henceforth named 'hypoxia-induced noncoding ultraconserved transcripts' (HINCUTs), are also overexpressed in clinical samples from colon cancer patients. We show that these T-UCRs are predominantly nuclear and that the hypoxia-inducible factor (HIF) is at least partly responsible for the induction of several members of this group. One specific HINCUT, uc.475 (or HINCUT-1) is part of a retained intron of the host protein-coding gene, O-linked N-acetylglucosamine transferase, which is overexpressed in epithelial cancer types. Consistent with the hypothesis that T-UCRs have important function in tumor formation, HINCUT-1 supports cell proliferation specifically under hypoxic conditions and may be critical for optimal O-GlcNAcylation of proteins when oxygen tension is limiting. Our data gives a first glimpse of a novel functional hypoxic network comprising protein-coding transcripts and noncoding RNAs (ncRNAs) from the T-UCRs category.

Control of FLIP(L) expression and TRAIL resistance by the extracellular signal-regulated kinase1/2 pathway in breast epithelial cells.

Increased activation of the epidermal growth factor receptor (EGFR) is frequently observed in tumors, and inhibition of the signaling pathways originated in the EGFR normally renders tumor cells more sensitive to apoptotic stimuli. However, we show that inhibition of EGFR signaling in non-transformed breast epithelial cells by EGF deprivation or gefitinib, an inhibitor of EGFR tyrosine kinase, causes the upregulation of the long isoform of caspase-8 inhibitor FLICE-inhibitory protein (FLIP(L)) and makes these cells more resistant to the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). We demonstrate that the extracellular signal-regulated kinase (ERK)1/2 pathway plays a pivotal role in the regulation of FLIP(L) levels and sensitivity to TRAIL-induced apoptosis by EGF. Upregulation of FLIP(L) upon EGF deprivation correlates with a decrease in c-Myc levels and c-Myc knockdown by siRNA induces FLIP(L) expression. FLIP(L) upregulation and resistance to TRAIL in EGF-deprived cells are reversed following activation of an estrogen activatable form of c-Myc (c-Myc-ER). Finally, constitutive activation of the ERK1/2 pathway in HER2/ERBB2-transformed cells prevents EGF deprivation-induced FLIP(L) upregulation and TRAIL resistance. Collectively, our results suggest that a regulated ERK1/2 pathway is crucial to control FLIP(L) levels and sensitivity to TRAIL in non-transformed cells, and this mechanism may explain the increased sensitivity of tumor cells to TRAIL, in which the ERK1/2 pathway is frequently deregulated.

Nutrient sensor O-GlcNAc transferase regulates breast cancer tumorigenesis through targeting of the oncogenic transcription factor FoxM1.

Cancer cells upregulate glycolysis, increasing glucose uptake to meet energy needs. A small fraction of a cell's glucose enters the hexosamine biosynthetic pathway (HBP), which regulates levels of O-linked beta-N-acetylglucosamine (O-GlcNAc), a carbohydrate posttranslational modification of diverse nuclear and cytosolic proteins. We discovered that breast cancer cells upregulate the HBP, including increased O-GlcNAcation and elevated expression of O-GlcNAc transferase (OGT), which is the enzyme catalyzing the addition of O-GlcNAc to proteins. Reduction of O-GlcNAcation through RNA interference of OGT in breast cancer cells leads to inhibition of tumor growth both in vitro and in vivo and is associated with decreased cell-cycle progression and increased expression of the cell-cycle inhibitor p27(Kip1). Elevation of p27(Kip1) was associated with decreased expression and activity of the oncogenic transcription factor FoxM1, a known regulator of p27(Kip1) stability through transcriptional control of Skp2. Reducing O-GlcNAc levels in breast cancer cells decreased levels of FoxM1 protein and caused a decrease in multiple FoxM1-specific targets, including Skp2. Moreover, reducing O-GlcNAcation decreased cancer cell invasion and was associated with the downregulation of matrix metalloproteinase-2, a known FoxM1 target. Finally, pharmacological inhibition of OGT in breast cancer cells had similar anti-growth and anti-invasion effects. These findings identify O-GlcNAc as a novel mechanism through which alterations in glucose metabolism regulate cancer growth and invasion and suggest that OGT may represent novel therapeutic targets for breast cancer.

Interdomain communication regulating ligand binding by PPAR-gamma.

Binding to receptors in the cell nucleus is crucial for the action of lipophilic hormones and ligands. PPAR-gamma (for peroxisome proliferator-activated receptor) is a nuclear hormone receptor that mediates adipocyte differentiation and modulates insulin sensitivity, cell proliferation and inflammatory processes. PPAR-gamma ligands have been implicated in the development of atherogenic foam cells and as potential cancer treatments. Transcriptional activity of PPAR-gamma is induced by binding diverse ligands, including natural fatty acid derivatives, antidiabetic thiazolidinediones, and non-steroidal anti-inflammatory drugs. Ligand binding by PPAR-gamma, as well as by the entire nuclear-receptor superfamily, is an independent property of the carboxy-terminal ligand-binding domain (LBD) of the receptor. Here we show that ligand binding by PPAR-gamma is regulated by intramolecular communication between its amino-terminal A/B domain and its carboxy-terminal LBD. Modification of the A/B domain, for example by physiological phosphorylation by MAP kinase, reduces ligand-binding affinity, thus negatively regulating the transcriptional and biological functions of PPAR-gamma. The ability of the A/B domain to regulate ligand binding has important implications for the evaluation and mechanism of action of potentially therapeutic ligands that bind PPAR-gamma and that are likely to extend to other members of the nuclear-receptor superfamily.

A potent antidiabetic thiazolidinedione with unique peroxisome proliferator-activated receptor gamma-activating properties.

Thiazolidinediones (TZDs) constitute an exciting new class of antidiabetic compounds, which function as activating ligands for peroxisome proliferator-activated receptor gamma (PPARgamma). Until now, there has been an excellent correlation between in vivo hypoglycemic potency and in vitro binding and activation of PPARgamma by TZDs. We have characterized MCC-555, a novel thiazolidinedione ligand for PPARgamma with unique functional properties. The antidiabetic potency of this compound is greater than that of other TZDs, including BRL49653, yet its binding affinity for PPARgamma is less than (1)/(10) that of BRL49653. The effect of MCC-555 binding on PPARgamma transcriptional activity is highly context-specific such that it can function as a full agonist, partial agonist, or antagonist depending on the cell type or DNA binding site. These transcriptional properties are partly explained by unique partial agonism of coactivator recruitment to PPARgamma. The properties of MCC-555 are mechanistically distinct from those of the estrogen receptor partial agonist and antagonist tamoxifen because the N terminus of PPARgamma is not required for activation by MCC-555, and MCC-555 does not stimulate corepressor recruitment to PPARgamma. The context selectivity of MCC-555 may contribute to its enhanced hypoglycemic potency in vivo despite reduced affinity for PPARgamma relative to other TZDs.

Prostaglandins promote and block adipogenesis through opposing effects on peroxisome proliferator-activated receptor gamma.

Fat cell differentiation is a critical aspect of obesity and diabetes. Dietary fatty acids are converted to arachidonic acid, which serves as precursor of prostaglandins (PGs). PGJ2 derivatives function as activating ligands for peroxisome proliferator-activated receptor gamma (PPAR gamma), a nuclear hormone receptor that is central to adipogenic determination. We report here that PGF2 alpha blocks adipogenesis through activation of mitogen-activated protein kinase, resulting in inhibitory phosphorylation of PPAR gamma. Both mitogen-activated protein kinase activation and PPAR gamma phosphorylation are required for the anti-adipogenic effects of PGF2 alpha. Thus, PG signals generated at a cell surface receptor regulate the program of gene expression required for adipogenesis by modulating the activity of a nuclear hormone receptor that is directly activated by other PG signals. The balance between PGF2 alpha and PGJ2 signaling may thus be central to the development of obesity and diabetes.

The transcriptional integrator CREB-binding protein mediates positive cross talk between nuclear hormone receptors and the hematopoietic bZip protein p45/NF-E2.

Thyroid hormone (T3) and retinoic acid (RA) play important roles in erythropoiesis. We found that the hematopoietic cell-specific bZip protein p45/NF-E2 interacts with T3 receptor (TR) and RA receptor (RAR) but not retinoid X receptor. The interaction is between the DNA-binding domain of the nuclear receptor and the leucine zipper region of p45/NF-E2 but is markedly enhanced by cognate ligand. Remarkably, ligand-dependent transactivation by TR and RAR is markedly potentiated by p45/NF-E2. This effect of p45/NF-E2 is prevented by maf-like protein p18, which functions positively as a heterodimer with p45/NF-E2 on DNA. Potentiation of hormone action by p45/NF-E2 requires its activation domain, which interacts strongly with the multifaceted coactivator cyclic AMP response element protein-binding protein (CBP). The region of CBP which interacts with p45/NF-E2 is the same interaction domain that mediates inhibition of hormone-stimulated transcription by AP1 transcription factors. Overexpression of the bZip interaction domain of CBP specifically abolishes the positive cross talk between TR and p45/NF-E2. Thus, positive cross talk between p45/NF-E2 and nuclear hormone receptors requires direct protein-protein interactions between these factors and with CBP, whose integration of positive signals from two transactivation domains provides a novel mechanism for potentiation of hormone action in hematopoietic cells.

Retinoic acid blocks adipogenesis by inhibiting C/EBPbeta-mediated transcription.

Adipocyte differentiation is thought to involve sequential induction of the transcription factors C/EBPbeta, peroxisome proliferator-activated receptor gamma (PPARgamma), and C/EBPalpha. C/EBPalpha expression is both necessary and sufficient for adipocyte differentiation. Here we report that ectopic expression of either C/EBPalpha or C/EBPbeta induces PPARgamma expression and adipogenesis and that retinoic acid (RA) completely inhibits adipogenesis by either form of C/EBP. In studies of normal preadipocytes, RA does not prevent C/EBPbeta induction but blocks induction of PPARgamma, C/EBPalpha, and adipogenesis. In transient transfection studies, liganded RA receptor (RAR) specifically blocks transcriptional activation by either C/EBPalpha or C/EBPbeta. These results strongly suggest that C/EBPalpha substitutes for C/EBPbeta to induce adipocyte differentiation and that liganded RAR inhibits adipogenesis by blocking C/EBPbeta-mediated induction of downstream genes.

Transcriptional activation by peroxisome proliferator-activated receptor gamma is inhibited by phosphorylation at a consensus mitogen-activated protein kinase site.

The nuclear receptor peroxisome proliferator-activated receptor gamma (PPARgamma) regulates transcription in response to prostanoid and thiazolidinedione ligands and promotes adipocyte differentiation. The amino-terminal A/B domain of this receptor contains a consensus mitogen-activated protein kinase site in a region common to PPARgamma1 and -gamma2 isoforms. The A/B domain of human PPARgamma1 was phosphorylated in vivo, and this was abolished either by mutation of serine 84 to alanine (S84A) or coexpression of a phosphoprotein phosphatase. In vitro, this domain was phosphorylated by ERK2 and JNK, and this was markedly reduced in the S84A mutant. A wild type Gal4-PPARgamma(A/B) chimera exhibited weak constitutive transcriptional activity. Remarkably, this was significantly enhanced in the S84A mutant fusion. Ligand-dependent activation by full-length mouse PPARgamma2 was also augmented by mutation of the homologous serine in the A/B domain to alanine. The nonphosphorylatable form of PPARgamma was also more adipogenic. Thus, phosphorylation of a mitogen-activated protein kinase site in the A/B region of PPARgamma inhibits both ligand-independent and ligand-dependent transactivation functions. This observation provides a potential mechanism whereby transcriptional activation by PPARgamma may be modulated by growth factor or cytokine-stimulated signal transduction pathways involved in adipogenesis.

DNA-independent and DNA-dependent mechanisms regulate the differential heterodimerization of the isoforms of the thyroid hormone receptor with retinoid X receptor.

Thyroid hormone receptors (TRs) require heterodimerization with retinoid X receptor (RXR) for maximum DNA binding affinity. Interaction with RXR occurs via two dimerization interfaces, one in the DNA-binding domain and one in the C-terminal "ninth heptad" of the receptors. We studied the relative importance of these two dimerization domains in naturally occurring C-terminal TR variants. TRalpha1 has a conserved ninth heptad and formed stable heterodimers with RXR in solution. TRalpha1.RXR heterodimers bound similarly to direct repeat 4 (DR4) sites with different 5'-flanking and spacer sequences. In contrast, TRalpha2, which contains a highly divergent ninth heptad, did not interact with RXR in solution and bound as a heterodimer with RXR only to specific DR4 sequences in which the downstream half-site was the preferred octameric binding site of TR (TNAGGTCA). Although the ninth heptad of TRalpha2 was insufficient for interaction with RXR off DNA, this region was required for DNA-dependent heterodimerization with RXR. TRalpha3, another naturally occurring TRalpha isoform whose ninth heptad differs from those of both TRalpha1 and TRalpha2, displayed intermediate behavior in heterodimerization with RXR. Thus, in the absence of a strong ninth heptad interaction an octameric downstream half-site allosterically promotes RXR heterodimerization with TRalpha2. Differential dependence upon DNA-binding for heterodimerization with RXR may influence transcriptional regulation by TRalpha isoforms.

Functional regulation of thyroid hormone receptor variant TR alpha 2 by phosphorylation.

The thyroid hormone (T3) receptor (TR) variant TR alpha 2 is abundant in brain but does not bind T3 because of its unique C terminus. The only known function of TR alpha 2, inhibition of TR-dependent transactivation, involves competition for T3 response elements. Paradoxically, in vitro-translated TR alpha 2 bound poorly to these sites. We report here that dephosphorylation of TR alpha 2 restored its DNA binding. Mutation of C-terminal serine residues to alanine (TR alpha 2-SA) was equally effective. The C terminus of TR alpha 2 was phosphorylated in a human cell line, whereas that of TR alpha 2-SA was not. Conversely, TR alpha 2-SA was a much better inhibitor of T3 action than was wild-type TR alpha 2. The dominant negative activity of TR alpha 2-SA was less than stoichiometric with TR concentration, possibly because it was unable to heterodimerize with retinoid X receptor, which enhances the binding of other TRs. Purified casein kinase II as well as a reticulocyte casein kinase II-like activity phosphorylated TR alpha 2 on serines 474 and 475. Mutation of these two residues to alanine was sufficient to restore DNA binding. Thus, DNA binding by TR alpha 2 is regulated by phosphorylation at a site distant from the DNA-binding domain. The increased dominant negative activity of a nonphosphorylatable form of TR alpha 2 suggests that phosphorylation may provide a rapid, T3-independent mechanism for cell-specific modulation of the expression of T3-responsive genes.

Hypotension induced by growth-hormone-releasing peptide is mediated by mast cell serotonin release in the rat.

Growth-hormone-releasing peptide (GH-RP-6) is a synthetic hexapeptide that selectively releases growth hormone (GH) when administered to a number of animals species. In the rat, maximal GH release occurs after intravenous administration of 100 micrograms/kg GH-RP-6. Intravenous administration of 5 mg/kg GH-RP-6 produced 100% lethality within 2-5 min of drug administration. Further investigative studies demonstrated that the lethal effect of GH-RP-6 was preceded by an initial hypertensive episode, followed by a rapid, profound hypotension and bradycardia. The rise and fall in blood pressure also were observed in pithed rats treated with GH-RP-6, suggesting that the central nervous system was not responsible for the changes in blood pressure. However, the GH-RP-6-induced bradycardia was not observed in pithed rats, indicating the fall in heart rate was mediated through a central reflex mechanism. No direct effects of GH-RP-6 were seen in the isolated rat aorta or canine saphenous vein. Pretreatment of conscious rats with naloxone (10 mg/kg, iv), an opiate receptor antagonist, did not prevent the hypertensive response to GH-RP-6, but the hypotension and lethality were attenuated. Pretreatment with cyproheptadine (2.5 mg/kg, iv), a dual serotonin/histamine antagonist, or ketanserin (3 mg/kg, iv), a selective serotonin antagonist, prevented the GH-RP-6-induced hypotension and lethality. Cyproheptadine unmasked a 40 mm Hg rise in mean arterial pressure which persisted for over 10 min. In addition, degranulation of mast cells with compound 48/80 inhibited the toxicity of GH-RP-6, suggesting that mast cell degranulation and the subsequent release of autocoids is responsible for the cardiovascular effects of GH-RP-6. In vitro, GH-RP-6 (10(-5) - 10(-3) M) produced a concentration-related release of histamine from rat peritoneal mast cells. However, the histamine release by GH-RP-6 (10(-4) M) was not inhibited by naloxone (10(-4) M) in isolated mast cells, suggesting either that peritoneal mast cells are not responsible or that the mast cell degranulation in vitro is not opiate mediated. In conclusion, it appears that GH-RP-6 degranulates mast cells releasing serotonin, which produces hypotension, bradycardia, and death. This degranulation of mast cells is apparently inhibited by naloxone in vivo, suggesting that opiate receptors are involved in the hypotension and lethality associated with the administration of GH-RP-6.