Valerie L. Sodi

O-GlcNAcylation Regulates Cancer Metabolism and Survival Stress Signaling via Regulation of the HIF-1 Pathway

The hexosamine biosynthetic pathway elevates posttranslational addition of O-linked β-N-acetylglucosamine (O-GlcNAc) on intracellular proteins. Cancer cells elevate total O-GlcNAcylation by increasing O-GlcNAc transferase (OGT) and/or decreasing O-GlcNAcase (OGA) levels. Reducing O-GlcNAcylation inhibits oncogenesis. Here, we demonstrate that O-GlcNAcylation regulates glycolysis in cancer cells via hypoxia-inducible factor 1 (HIF-1α) and its transcriptional target GLUT1. Reducing O-GlcNAcylation increases α-ketoglutarate, HIF-1 hydroxylation, and interaction with von Hippel-Lindau protein (pVHL), resulting in HIF-1α degradation. Reducing O-GlcNAcylation in cancer cells results in activation of endoplasmic reticulum (ER) stress and cancer cell apoptosis mediated through C/EBP homologous protein (CHOP). HIF-1α and GLUT1 are critical for OGT-mediated regulation of metabolic stress, as overexpression of stable HIF-1 or GLUT1 rescues metabolic defects. Human breast cancers with high levels of HIF-1α contain elevated OGT, and lower OGA levels correlate independently with poor patient outcome. Thus, O-GlcNAcylation regulates cancer cell metabolic reprograming and survival stress signaling via regulation of HIF-1α.

O-GlcNAcylation in Cancer Biology: Linking Metabolism and Signaling

The hexosamine biosynthetic pathway (HBP) is highly dependent on multiple metabolic nutrients including glucose, glutamine, and acetyl-CoA. Increased flux through HBP leads to elevated post-translational addition of β-D-N-acetylglucosamine sugars to nuclear and cytoplasmic proteins. Increased total O-GlcNAcylation is emerging as a general characteristic of cancer cells, and recent studies suggest that O-GlcNAcylation is a central communicator of nutritional status to control key signaling and metabolic pathways that regulate multiple cancer cell phenotypes. This review summarizes our current understanding of changes of O-GlcNAc cycling enzymes in cancer, the role of O-GlcNAcylation in tumorigenesis, and the current challenges in targeting this pathway therapeutically.

mTOR/MYC Axis Regulates O-GlcNAc Transferase Expression and O-GlcNAcylation in Breast Cancer

Cancers exhibit altered metabolism characterized by increased glucose and glutamine uptake. The hexosamine biosynthetic pathway (HBP) uses glucose and glutamine, and directly contributes to O-linked-β-N-acetylglucosamine (O-GlcNAc) modifications on intracellular proteins. Multiple tumor types contain elevated total O-GlcNAcylation, in part, by increasing O-GlcNAc transferase (OGT) levels, the enzyme that catalyzes this modification. Although cancer cells require OGT for oncogenesis, it is not clear how tumor cells regulate OGT expression and O-GlcNAcylation. Here, it is shown that the PI3K–mTOR–MYC signaling pathway is required for elevation of OGT and O-GlcNAcylation in breast cancer cells. Treatment with PI3K and mTOR inhibitors reduced OGT protein expression and decreased levels of overall O-GlcNAcylation. In addition, both AKT and mTOR activation is sufficient to elevate OGT/O-GlcNAcylation. Downstream of mTOR, the oncogenic transcription factor c-MYC is required and sufficient for increased OGT protein expression in an RNA-independent manner and c-MYC regulation of OGT mechanistically requires the expression of c-MYC transcriptional target HSP90A. Finally, mammary tumor epithelial cells derived from MMTV-c-myc transgenic mice contain elevated OGT and O-GlcNAcylation and OGT inhibition in this model induces apoptosis. Thus, OGT and O-GlcNAcylation levels are elevated via activation of an mTOR/MYC cascade. Implications: Evidence indicates OGT as a therapeutic target in c-MYC–amplified cancers. Mol Cancer Res; 13(5); 923–33. ©2015 AACR.

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.

Light-dependent expression of four cryptic archaeal circadian gene homologs.

Circadian rhythms are important biological signals that have been found in almost all major groups of life from bacteria to man, yet it remains unclear if any members of the second major prokaryotic domain of life, the Archaea, also possess a biological clock. As an initial investigation of this question, we examined the regulation of four cyanobacterial-like circadian gene homologs present in the genome of the haloarchaeon Haloferax volcanii. These genes, designated cirA, cirB, cirC, and cirD, display similarity to the KaiC-family of cyanobacterial clock proteins, which act to regulate rhythmic gene expression and to control the timing of cell division. Quantitative RT-PCR analysis was used to examine the expression of each of the four cir genes in response to 12 h light/12 h dark cycles (LD 12:12) in H. volcanii during balanced growth. Our data reveal that there is an approximately two to sixteen-fold increase in cir gene expression when cells are shifted from light to constant darkness, and this pattern of gene expression oscillates with the light conditions in a rhythmic manner. Targeted single- and double-gene knockouts in the H. volcanii cir genes result in disruption of light-dependent, rhythmic gene expression, although it does not lead to any significant effect on growth under these conditions. Restoration of light-dependent, rhythmic gene expression was demonstrated by introducing, in trans, a wild-type copy of individual cir genes into knockout strains. These results are noteworthy as this is the first attempt to characterize the transcriptional expression and regulation of the ubiquitous kaiC homologs found among archaeal genomes.

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.

mTOR/MYC Axis Regulates O-GlcNAc Transferase (OGT) Expression and O-GlcNAcylation in Breast Cancer

Cancers exhibit altered metabolism characterized by increased glucose and glutamine uptake. The hexosamine biosynthetic pathway (HBP) uses glucose and glutamine, and directly contributes to O-linked-β-N-acetylglucosamine (O-GlcNAc) modifications on intracellular proteins. Multiple tumor types contain elevated total O-GlcNAcylation, in part, by increasing O-GlcNAc transferase (OGT) levels, the enzyme that catalyzes this modification. Although cancer cells require OGT for oncogenesis, it is not clear how tumor cells regulate OGT expression and O-GlcNAcylation. Here, it is shown that the PI3K–mTOR–MYC signaling pathway is required for elevation of OGT and O-GlcNAcylation in breast cancer cells. Treatment with PI3K and mTOR inhibitors reduced OGT protein expression and decreased levels of overall O-GlcNAcylation. In addition, both AKT and mTOR activation is sufficient to elevate OGT/O-GlcNAcylation. Downstream of mTOR, the oncogenic transcription factor c-MYC is required and sufficient for increased OGT protein expression in an RNA-independent manner and c-MYC regulation of OGT mechanistically requires the expression of c-MYC transcriptional target HSP90A. Finally, mammary tumor epithelial cells derived from MMTV-c-myc transgenic mice contain elevated OGT and O-GlcNAcylation and OGT inhibition in this model induces apoptosis. Thus, OGT and O-GlcNAcylation levels are elevated via activation of an mTOR/MYC cascade. Implications: Evidence indicates OGT as a therapeutic target in c-MYC–amplified cancers. Mol Cancer Res; 13(5); 923–33. ©2015 AACR.

Population Structure of Adult Blue Crabs, Callinectes sapidus, in Relation to Physical Characteristics in Barnegat Bay, New Jersey

Blue crabs (Callinectes sapidus) are an important species in coastal or lagoonal estuaries where adult population characteristics may differ as compared to drowned-river estuaries. Barnegat Bay, in southern New Jersey, is composed of two large embayments: one without and one with a salinity gradient. We tested the influence of physical characteristics on the abundance, sex ratio, and size of adult blue crabs and examined variation in measures of reproductive potential (e.g., sperm stores) in both sexes in Barnegat Bay from June to September, 2008–2009. Population structure was distinct between the embayments due to sex-specific responses to salinity: male abundance was negatively correlated with salinity whereas adult females were more abundant in high salinity because of proximity to Barnegat Inlet. This produced high sex ratios in low salinity areas and low sex ratios in high salinity areas. Summer was a growing season for adult males while in late summer-early fall, juvenile males recruited to the adult size class. The spawning season lasted from May to August and ovigerous females were concentrated near the inlets. Information on female sperm stores and ovarian development identified two cohorts of adult females: females that will spawn in the current summer and females that will not spawn until the following summer. Thus, not all adult females near the spawning grounds were members of the current spawning stock. This suggests that annual estimates of spawning stock size which overlook the proximity of females to spawning are overestimating the current spawning stock in Barnegat Bay and other estuaries.

MTOR/MYCAxis regulates O-GlcNAc transferase expression and O-GlcN acylation in breast cancer

Cancers exhibit altered metabolism characterized by increased glucose and glutamine uptake. The hexosamine biosynthetic pathway (HBP) uses glucose and glutamine, and directly contributes to O-linked-β-N-acetylglucosamine (O-GlcNAc) modifications on intracellular proteins. Multiple tumor types contain elevated total O-GlcNAcylation, in part, by increasing O-GlcNAc transferase (OGT) levels, the enzyme that catalyzes this modification. Although cancer cells require OGT for oncogenesis, it is not clear how tumor cells regulate OGT expression and O-GlcNAcylation. Here, it is shown that the PI3K–mTOR–MYC signaling pathway is required for elevation of OGT and O-GlcNAcylation in breast cancer cells. Treatment with PI3K and mTOR inhibitors reduced OGT protein expression and decreased levels of overall O-GlcNAcylation. In addition, both AKT and mTOR activation is sufficient to elevate OGT/O-GlcNAcylation. Downstream of mTOR, the oncogenic transcription factor c-MYC is required and sufficient for increased OGT protein expression in an RNA-independent manner and c-MYC regulation of OGT mechanistically requires the expression of c-MYC transcriptional target HSP90A. Finally, mammary tumor epithelial cells derived from MMTV-c-myc transgenic mice contain elevated OGT and O-GlcNAcylation and OGT inhibition in this model induces apoptosis. Thus, OGT and O-GlcNAcylation levels are elevated via activation of an mTOR/MYC cascade. Implications: Evidence indicates OGT as a therapeutic target in c-MYC–amplified cancers. Mol Cancer Res; 13(5); 923–33. ©2015 AACR.

Abstract 1194: O-GlcNAcylation regulates breast cancer lipid metabolism via sterol regulatory element binding protein 1

The Warburg hypothesis states that cancer cells display altered metabolic features to support their rapid growth and biosynthetic demands including increased glycolytic flux to synthesize necessary building blocks required by growing cells. Increased glucose uptake feeds not only glycolysis but other glucose dependent pathways as well, such as the Hexosamine Biosynthetic Pathway (HBP). HBP diverts fructose-6-phosphate from glycolysis to produce UDP-GlcNAc, the amino sugar donor to O-GlcNAc Transferase (OGT). Analagous to phosphorylation, O-GlcNAcylation can alter protein function and stability and itself is altered in several disease states. Our lab has demonstrated that OGT and O-GlcNAcylation are elevated in breast cancer and that expression of this enzyme is critical for various cancer phenotypes such as avoidance of ER stress and apoptosis, supporting invasion and metastasis and more recently promotion of Warburg effect. Here we demonstrate that OGT and O-GlcNAc are important for lipid metabolism in cancer cells through regulation of transcription factor SREBP-1 and its targets. Metabolic profiling revealed that OGT depletion in MDA-MB-231 cells decreased various lipids metabolites measured, including long-chain fatty acids, lysolipids and essential fatty acids. OGT suppression results in a decrease in SREBP-1 protein and mRNA and reduction of its transcriptional targets, enzymes involved in lipid synthesis, in both breast cancer cells and highly lipogenic normal tissue, the mammary fat pad. Over-expression of SREBP-1 blocks apoptosis induced by OGT suppression in 2D and 3D culture. Overall, we show that OGT and O-GlcNAc play a role in yet another process critical to cancer cell survival, lipid metabolism. Citation Format: Valerie L. Sodi, Zachary Bacigalupa, Christina Ferrer, Mauricio Reginato. O-GlcNAcylation regulates breast cancer lipid metabolism via sterol regulatory element binding protein 1. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1194. doi:10.1158/1538-7445.AM2015-1194