The Nrf1 (Nuclear factor E2-related factor 1) transcription factor performs a critical role in regulating cellular homeostasis. Using a proteomic approach, we identified Host Cell Factor-1 (HCF1), a co-regulator of transcription, and O-GlcNAc transferase (OGT), the enzyme that mediates protein O-GlcNAcylation, as cellular partners of Nrf1a, an isoform of Nrf1. Nrf1a directly interacts with HCF1 through the HCF1 binding motif (HBM), while interaction with OGT is mediated through HCF1. Overexpression of HCF1 and OGT leads to increased Nrf1a protein stability. Addition of O-GlcNAc decreases ubiquitination and degradation of Nrf1a. Transcriptional activation by Nrf1a is increased by OGT overexpression and treatment with PUGNAc. Together, these data suggest that OGT can act as a regulator of Nrf1a.

Pancreatic cancer is one of the most aggressive malignancies that is often diagnosed at a late stage following tumor metastasis. Given the low response rate to current therapies, there is a clear and urgent need for new therapeutic and diagnostic approaches. Proteomics is an emerging tool that offers the opportunity to discover novel antigens elevated in cancer. Considering the large number of marketed drugs that target plasma membrane proteins, and given their amenability to both small molecule and antibody-based technologies, an approach that combines a cell membrane labeling strategy with proteomic (LC-MS/MS) methodology would enable novel cell surface antigens that can act as novel biomarkers and/or targets for drug development to be identified. One major consideration when selecting targets for therapeutics is whether or not the antigen plays a functional role in a disease context. RNA interference (RNAi) permits the downregulation of any given gene, and therefore allows the identification of functionality in the target disease. As such, this tool permits the selection of targets prior to investment in a therapeutic antibody program or an extensive small molecule–screening program. In this chapter, we describe the utility of the cell membrane labeling strategy and LC-MS/MS method that was developed at Celera. Subsequent application of RNAi allowed us to assign functionality to cell surface antigens we identified that were upregulated in pancreatic cancer. By using a combination of a proteomics and an RNAi platform, we demonstrate that it is possible to discover novel therapeutic and diagnostic targets for this devastating disease.

The ubiquitin-proteasome system is important in maintaining protein homeostasis. NFE2-related factor 1 (Nrf1), a transcription factor in the cap n' collar basic-leucine zipper family, regulates expression of cytoprotective genes. It was previously shown that liver-specific knockout of Nrf1 (Nrf1LKO) leads to hepatic cell death, steatohepatitis and cancer. However, the mechanisms underlying these pathologies are not clear. Here, we report that Nrf1 is critical for proteasome gene expression in the liver. Liver-specific knockout of Nrf1 results in impaired basal and induced expression of proteasome genes, and diminished proteasome activity in hepatocytes. In addition, our findings demonstrated that endoplasmic reticulum stress signaling pathway was also activated in Nrf1LKO livers. Inhibition of proteasome activity leads to endoplasmic reticulum stress in Nrf1-deficient hepatocytes, prompting the development of steatosis in the liver. Our results indicate that Nrf1 plays an integral role in the maintenance of proteasome function in hepatocytes and in the prevention of liver steatosis development. Moreover, these results highlight an association between proteasome dysfunction, endoplasmic reticulum stress and steatosis.

Objectives: There is a clear need for better therapeutics and diagnostics for pancreatic cancer. We aimed to discover plasma membrane-associated proteins overexpressed in pancreatic cancer using quantitative proteomics and apply RNA interference (RNAi) to uncover proteins associated with cancer cell survival.Methods: Cell surface glycoproteins from 5 pancreatic cancer cell lines were isolated, and differential analyses were performed using mass spectrometry and the "normoid" cell line Hs766T as the comparator. For validation, immunohistochemistry was performed on tissues from 10 independent patients and 2 normal donors. Correlation of protein and mRNA expression level was determined, and functional activity characterized using RNAi.Results: Integrin beta 6, CD46, tissue factor, and a novel protein, chromosome 14 open reading frame 1, were identified as overexpressed on pancreatic cancer cell lines. Immunohistochemistry demonstrated the 4 targets were overexpressed in 20% to 70% of primary pancreatic tumor specimens. Small interfering RNA knockdown resulted in a reduction of cellular proliferation by inhibiting DNA synthesis, blocking S-phase progression or induction of apoptosis.Conclusions: By combining a mass spectrometry identification platform and an RNAi validation platform, we have identified a panel of cell surface glycoproteins that not only are overexpressed, but also play a functional role in pancreatic tumor cell survival.

The ubiquitin–proteasome pathway plays an important role in the pathogenesis of neurodegeneration, but mechanisms controlling expression of components in this pathway remain poorly understood. Nuclear factor E2-related factor 1 (Nrf1) transcription factor has been shown to regulate expression of antioxidant and cytoprotective genes. To determine the function of Nrf1 in the brain, mice with a late-stage deletion of Nrf1 in neuronal cells were generated. Loss of Nrf1 leads to impaired proteasome function and neurodegeneration. Gene expression profiling and RT-PCR analysis revealed a coordinate down-regulation of various proteasomal genes including PsmB6, which encodes a catalytic subunit of the proteasome. Transcriptional analysis and chromatin immunoprecipitation experiments demonstrated that PsmB6 is an Nrf1 target gene. These findings reveal Nrf1 as a key transcriptional regulator required for the expression of proteasomal genes in neurons and suggest that perturbations of Nrf1 function may contribute to the pathogenesis of neurodegenerative diseases.

In Saccharomyces cerevisiae, chemical or genetic inhibition of proteasome activity induces new proteasome synthesis promoted by the transcription factor RPN4. This ensures that proteasome activity is matched to demand. This transcriptional feedback loop is conserved in mammals, but its molecular basis is not understood. Here, we report that nuclear factor erythroid-derived 2-related factor 1 (Nrf1), a transcription factor of the cap "n" collar basic leucine zipper family, but not the related Nrf2, is necessary for induced proteasome gene transcription in mouse embryonic fibroblasts (MEFs). Promoter-reporter assays revealed the importance of antioxidant response elements in Nrf1-mediated upregulation of proteasome subunit genes. Nrf1(-/-) MEFs were impaired in the recovery of proteasome activity after transient treatment with the covalent proteasome inhibitor YU101, and knockdown of Nrf1 in human cancer cells enhanced cell killing by YU101. Taken together, our results suggest that Nrf1-mediated proteasome homeostasis could be an attractive target for therapeutic intervention in cancer.

Nuclear factor E2-related factor 2 (Nrf2) is a cap-n-collar basic leucine zipper (CNC-bZIP) transcription factor that is well established as a master regulator of phase II detoxification and antioxidant gene expression and is strongly expressed in tissues involved in xenobiotic metabolism including liver and kidney. Nrf2 is also abundantly expressed in adipose tissue; however, the exact function of Nrf2 in adipocyte biology is unclear. In the current study we show that targeted knock-out of Nrf2 in mice decreases adipose tissue mass, promotes formation of small adipocytes, and protects against weight gain and obesity otherwise induced by a high fat diet. In mouse embryonic fibroblasts, 3T3-L1 cells, and human subcutaneous preadipocytes, selective deficiency of Nrf2 impairs adipocyte differentiation. Deficiency of Nrf2 also leads to decreased expression of peroxisome proliferator-activated receptor gamma (PPAR gamma), CCAAT enhancer-binding protein alpha (C/EBP alpha), and their downstream targets during adipocyte differentiation. Conversely, activation of Nrf2 in 3T3-L1 cells by stable knockdown of its negative regulator Keap1 enhances and accelerates hormone-induced adipocyte differentiation. Transfection of Nrf2 stimulates Ppar gamma promoter activity, and stable knockdown of Keap1 enhances PPAR gamma expression in 3T3-L1 cells. In addition, chromatin immunoprecipitation studies show that Nrf2 associates with consensus binding sites for Nrf2 in the Ppar gamma promoter. These findings demonstrate a novel biologic role for Nrf2 beyond its participation in detoxification and antioxidant pathways and place Nrf2 within the limited network of transcription factors that control adipocyte differentiation by regulating expression of PPAR gamma.

Neurodegenerative disease is an important public health issue as the aged population continues to increase. Neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease and amyotrophic lateral sclerosis are caused by gradual loss of neurons. Although molecular mechanisms involved are not fully understood, accumulation of abnormal proteins, mitochondrial defects, and oxidative stress are common in many forms of neurodegenerative diseases. In order to cope with high levels of reactive oxygen species that are normally generated in the brain, antioxidant enzymes are transcriptionally activated in neurons to prevent oxidant damage. Members of CNC‐bZIP (Cap'n'Collar basic leucine zipper) transcription factor family have been implicated in regulating antioxidant gene expression through the antioxidant response element (ARE). Nrf1 is a CNC‐bZIP that is highly expressed in the adult brain, but its role in neurons is not known due to embryonic lethality in Nrf1 germ line mutant mice. Here, we generate and analyze Nrf1 brain conditional knockout mice by using Cre‐Lox system. Mice deficient of Nrf1 in the brain showed age dependent neurobehavioral abnormalities including abnormal leg‐clasping reflex, impaired rotarod performance and hyperactivity. Correspondingly, loss of Nrf1 in the mouse brain leads to age‐dependent brain atrophy as a result of apoptosis. Our studies here demonstrate a novel function of Nrf1 in protecting against neuronal apoptosis. Nrf1 Brain knockout mice may provide a model to study mechanisms of neurodegenerative diseases.

A99 Increasing evidence suggests that tumors depend on a subpopulation of cells termed cancer stem cells (CSCs) for tumor initiation and resistance to therapeutic intervention. Targeting the elimination of cancer stem cells therefore presents an opportunity for the development of more effective cancer treatments. A significant obstacle to characterizing cancer stem cells however is the limited ability to expand sufficient clonal populations for phenotypic characterization. Previously it has been demonstrated that CSC populations can be isolated and expanded as 3D tumorospheres from glioblastoma (Bao et al., Nature. 2006; 444:756) and melanoma (Fang et al., Cancer Research, 2005; 65:9328). More recently, CSC populations from colon carcinoma have been isolated based on their expression of the stem cell marker CD133 and expanded as 3D tumorospheres (Ricci-Vitiani et al., Nature 2007; 445:106). Here, we independently demonstrate isolation of tumorospheres from nine out of thirteen surgically resected colon tumor specimens of varying pathology stage and confirm that spheroid formation resides within the CD133 expressing population. Prolonged culturing of tumorospheres in an undifferentiated state was achieved for 3 independent colon cancer specimens with one in culture for over 22 months showing no notable senescence but retaining the ability to self-renew and differentiate towards an adherent epitheloid morphology. The long term spheroid cultures grow in an anchorage independent manner indicating their tumorogenic capacity. Expression of the stem cell markers nestin, BMI-1 and mushahsi-1 support their use as a model for stem cells. Significantly, they also displayed resistance to the growth inhibititory and apoptotic effects mediated by the chemotherapeutic irinotecan. To further characterize the tumorospheres, cell surface proteins were captured and subjected to LC-MS based quantitative proteomic analyses revealing enriched expression of CD133 and CD44. Taken together the characteristics of the expanded CD133 expressing colon cancer tumorospheres isolated suggest they have the potential to serve as a novel model system in which to identify and characterize treatments for colorectal cancer that are capable of targeting cancer stem cells.

B173 Prostate cancer is the second leading cause of cancer death in American men. The growing number of elderly people across the major markets will expand the overall prostate cancer incident population in the coming years. Identifying novel targets by proteomics may lead to opportunities for the development of targeted therapies or biomarkers. Additionally a significant clinical unmet need for prostate cancer is an effective drug therapy that is able to prevent the disease from progressing to androgen-refractory status or that can prolong the survival of patients with androgen-refractory disease. By employing mass spectrometry (MS) as a tool to identify proteins that are over-expressed in prostate tumor relative to normal prostate cells, we aimed to discover new targets that could be utilized in prostate cancer therapy. We developed proteomic methods that allowed us to focus our studies on the discovery of cell surface/secreted proteins, as they represent key antibody therapeutic and biomarker opportunities. Cell-surface and secreted proteins from normal and prostate tumor cell lines were preferentially captured, digested with trypsin and subjected to MS analysis. Peptides were first quantified, and then sequence composition of differentially expressed peptides was resolved by MS analysis. To date, we have identified in excess of 200 proteins over-expressed in prostate cancer cell lines, including known markers such as PSA, PSMA and a panel of proteins not previously associated with prostate cancer. Our analysis also showed 23 of identified proteins were uniquely found in prostate with no overlap with other oncology indications. Further analyses identified 37 proteins as over-expressed in androgen-refractory cell lines compared to androgen sensitive cell lines. Independently, a mouse xenograft study of androgen-dependent 22Rv1 prostate cell line was performed to investigate modulation of prostate tumor proteome expression upon androgen withdrawal. Xenografts from pre-castrated or re-grown post-castrated androgen-independent phase of the study were compared. Initial studies identified 21 proteins as over-expressed in 22Rv1 xenografts derived from post-castrated androgen-independent mice compared to pre-castrated mice. To validate proteins identified by MS, we performed additional confirmatory studies including immunohistochemistry (IHC), mRNA profiling, Flow Cytometry and functional analyses using RNAi-mediated transfection to evaluate the effect on prostate cell proliferation and/or apoptosis. A subset of targets was selected for validation based on criteria such as druggability of the proteins, novelty, and intensity of tumor over-expression. From this subset, 15 targets confirmed over-expression by IHC in prostate cancer tissues while 16 targets demonstrated RNAi function in prostate cell lines. Examples of such validated targets will be presented. These studies highlight that our large scale proteomic mapping capabilities can provide a platform for identification of novel therapeutics and biomarkers. Together with additional functional and expression characterization, this approach represents a unique opportunity for the discovery of targets that may be exploited in the diagnosis and treatment of prostate cancer.

a-Lipoic acid (LA), a potent antioxidant and free radical scavenger, has been reported to have insulin-sensitizing effects. In an attempt to improve the efficacy and antioxidant activity of LA, we synthesized and tested an ester derivative thereof: 6,8-bisacetylsulfanyloctanoic acid ethyl ester (LA-ester). Both LA and LA-ester were shown to be weak dual PPARa/g agonists, and to modulate the expression of PPAR-regulated genes, promote adipogenesis, and reduce cellular oxidative stress. Of interest, LA-ester markedly increased the maximum activity of both PPARa and PPARg, indicating increased efficacy. Both LA and LA-ester increased the expression of known PPAR a-regulated target genes (carnitine palmitoyltransferase 1A and acyl-CoA synthase) and PPARg-regulated target genes (fatty acid translocase/CD36, adipocyte fatty acidbinding protein, lipoprotein lipase) all of which are key regulators of lipid and glucose metabolism. LA-ester had a markedly higher capacity for reducing a-Lipoic Acid is a Weak Dual PPARa/g Agonist An Ester Derivative with Increased PPARa/g Efficacy and Antioxidant Activity Harrihar A. Pershadsingh, MD, PhD†* Christopher I. Ho‡ Jaya Rajamani‡ Candy Lee, PhD§ Amar G. Chittiboyina, PhDII Renu Deshpande, MD* Theodore W. Kurtz, MD# Jefferson Y. Chan, MD, PhD§ Mitchell A. Avery, PhD§ Stephen C. Benson, PhD‡ *Department of Family Medicine, Kern Medical Center, Bakersfield, California †Department of Family Medicine, University of California, Irvine, California ‡Department of Biological Sciences, California State University, Hayward, California §Department of Pathology, University of California, Irvine, California IIDepartment of Medicinal Chemistry, University of Mississippi, University, Mississippi #Department of Laboratory Medicine, University of California, San Francisco, California 16Pershadsingh-vol5no4 12/19/05 7:37 PM Page 510

Knockout studies have shown that the transcription factor Nrf1 is essential for embryonic development. Nrf1 has been implicated to play a role in mediating activation of oxidative stress response genes through the antioxidant response element (ARE). Because of embryonic lethality in knockout mice, analysis of this function in the adult knockout mouse was not possible. We report here that mice with somatic inactivation of nrf1 in the liver developed hepatic cancer. Before cancer development, mutant livers exhibited steatosis, apoptosis, necrosis, inflammation, and fibrosis. In addition, hepatocytes lacking Nrf1 showed oxidative stress, and gene expression analysis showed decreased expression of various ARE-containing genes, and up-regulation of CYP4A genes. These results suggest that reactive oxygen species generated from CYP4A-mediated fatty acid oxidation work synergistically with diminished expression of ARE-responsive genes to cause oxidative stress in mutant hepatocytes. Thus, Nrf1 has a protective function against oxidative stress and, potentially, a function in lipid homeostasis in the liver. Because the phenotype is similar to nonalcoholic steatohepatitis, these animals may prove useful as a model for investigating molecular mechanisms of nonalcoholic steatohepatitis and liver cancer.