O-GlcNAc transferase is critical for transducin-like enhancer of split (TLE)-mediated repression of canonical Wnt signaling.

The Drosophila Groucho protein and its mammalian orthologues the transducin-like enhancers of split (TLEs) are critical transcriptional corepressors that repress Wnt and other signaling pathways. Although it is known that Groucho/TLEs are recruited to target genes by pathway-specific transcription factors, molecular events after the corepressor recruitment are largely unclear. We report that association of TLEs with O-GlcNAc transferase, an enzyme that catalyzes posttranslational modification of proteins by O-linked N-acetylglucosamine, is essential for TLE-mediated transcriptional repression. Removal of O-GlcNAc from Wnt-responsive gene promoters is critical for gene activation from Wnt-responsive promoters. Thus, these studies identify a molecular mechanism by which Groucho/TLEs repress gene transcription and provide a model whereby O-GlcNAc may control distinct intracellular signaling pathways.

Muscle-specific overexpression of NCOATGK, splice variant of O-GlcNAcase, induces skeletal muscle atrophy.

The protein O-linked ß-N-acetylglucosamine (O-GlcNAc) modification plays an important role in skeletal muscle development and physiological function. In this study, bitransgenic mice were generated that overexpressed NCOAT(GK), an O-GlcNAcase-inactive spliced variant of the O-GlcNAcase gene, specifically in skeletal muscle using the muscle creatine kinase promoter. Expression of the chimeric enhanced green fluorescent protein-NCOAT(GK) transgene caused an increase of cellular O-GlcNAc levels, along with the accumulation and activation of proapoptotic factors in muscles of bitransgenic mice. The consequence of overexpressing the transgene for a 2-wk period was muscle atrophy and, in some cases, resulted in the death of male mice. Muscle atrophy is a common complication of many diseases, some of which correlate markedly with high cellular O-GlcNAc levels, such as diabetes. Our study provides direct evidence linking muscle atrophy and the disruption of O-GlcNAcase activity.

Identification of molecular distinctions between normal breast-associated fibroblasts and breast cancer-associated fibroblasts.

Stromal fibroblasts influence the behavior of breast epithelial cells. Fibroblasts derived from normal breast (NAF) inhibit epithelial growth, whereas fibroblasts from breast carcinomas (CAF) have less growth inhibitory capacity and can promote epithelial growth. We sought to identify molecules that are differentially expressed in NAF versus CAF and potentially responsible for their different growth regulatory abilities. To determine the contribution of soluble molecules to fibroblast-epithelial interactions, NAF were grown in 3D, transwell or direct contact co-cultures with MCF10AT epithelial cells. NAF suppressed proliferation of MCF10AT in both direct contact and transwell co-cultures, but this suppression was significantly greater in direct co-cultures, indicating involvement of both soluble and contact factors. Gene expression profiling of early passage fibroblast cultures identified 420 genes that were differentially expressed in NAF versus CAF. Of the eight genes selected for validation by real-time PCR, FIBULIN 1, was overexpressed in NAF, and DICKKOPF 1, NEUREGULIN 1, PLASMINOGEN ACTIVATOR INHIBITOR 2, and TISSUE PLASMINOGEN ACTIVATOR were overexpressed in CAF. A higher expression of FIBULIN 1 in normal- than cancer-associated fibroblastic stroma was confirmed by immunohistochemistry of breast tissues. Among breast cancers, stromal expression of Fibulin 1 protein was higher in estrogen receptor alpha-positive cancers and low stromal expression of Fibulin 1 correlated with a higher proliferation of cancer epithelial cells. In conclusion, expression profiling of NAF and CAF cultures identified many genes with potential relevance to fibroblast-epithelial interactions in breast cancer. Furthermore, these early passage fibroblast cultures can be representative of gene expression in stromal fibroblasts in vivo.

Human breast fibroblasts inhibit growth of the MCF10AT xenograft model of proliferative breast disease.

Stromal fibroblasts are important for normal breast homeostasis and regulation of epithelial growth; however, this regulatory function is altered during carcinogenesis. To study the role of fibroblasts in the development of breast cancer, fibroblasts derived from normal breast (NAFs) were incorporated into the MCF10AT xenograft model of progressive proliferative breast disease. The persistence of human NAFs in xenografts was established by intracellular labeling and tyramide-coupled fluorescent in situ hybridization. Overall, the number of MCF10AT epithelial structures was decreased, and the rate of epithelial cell apoptosis was increased in xenografts containing NAFs. However, these changes were primarily in low-grade epithelial structures, corresponding to normal or mildly hyperplastic ductal epithelium. The level and rate of apoptosis of high-grade epithelial structures, corresponding to in situ and invasive carcinoma, were not consistently altered by NAFs. In addition, there was variability in the growth-inhibitory capacity of NAFs derived from different individuals. NAFs induced changes in the morphology of high-grade MCF10AT structures and in xenograft stroma, including the composition of extracellular matrix, and increased angiogenesis and lymphocytic infiltration. These findings imply that NAFs can inhibit the growth of normal and hyperplastic epi-thelium but are less able to regulate the more transformed epithelial cells that arise during carcino-genesis.

O-GlcNAc integrates the proteasome and transcriptome to regulate nuclear hormone receptors.

Mechanisms controlling nuclear hormone receptors are a central question to mammalian developmental and disease processes. Herein, we show that a subtle increase in O-GlcNAc levels inhibits activation of nuclear hormone receptors. In vivo, increased levels of O-GlcNAc impair estrogen receptor activation and cause a decrease in mammary ductal side-branching morphogenesis associated with loss of progesterone receptors. Increased O-GlcNAc levels suppress transcriptional expression of coactivators and of the nuclear hormone receptors themselves. Surprisingly, increased O-GlcNAc levels are also associated with increased transcription of genes encoding corepressor proteins NCoR and SMRT. The association of the enzyme O-GlcNAc transferase with these corepressors contributes to specific regulation of nuclear hormone receptors by O-GlcNAc. Overall, transcriptional inhibition is related to the integrated effect of O-GlcNAc by direct modification of critical elements of the transcriptome and indirectly through O-GlcNAc modification of the proteasome.

Disrupting the enzyme complex regulating O-GlcNAcylation blocks signaling and development.

Although the knowledge that nuclear and cytoplasmic proteins are modified with N-acetylglucosamine has existed for decades, little has been shown as to its function until recently. There are now substantial data highlighting the significance of proper regulation of this modification in multiple cellular processes. Currently, only two enzymes are known that regulate this modification. O-GlcNAc transferase (OGT) modifies protein substrates posttranslationally by adding the N-acetylglucosamine. Bifunctional nuclear/cytoplasmic O-GlcNAcase and acetyl transferase (NCOAT) is responsible for cleaving the modification from target proteins. Here, we demonstrate for the first time an unusual association of these two opposing enzymes into a single O-GlcNAczyme complex. NCOAT and OGT associate strongly through specific domains such that NCOAT accompanies OGT, with histone deacetylases (HDACs), into transcription corepression complexes. Exclusion of NCOAT activities from OGT association blocks proper estrogen-dependent cell signaling as well as mammary development in transgenic mice. This demonstrates that NCOAT is in a strategic position to rapidly counteract OGT and HDAC without requiring its recruitment.

Breast fibroblasts modulate epithelial cell proliferation in three-dimensional in vitro co-culture.

BACKGROUND: Stromal fibroblasts associated with in situ and invasive breast carcinoma differ phenotypically from fibroblasts associated with normal breast epithelium, and these alterations in carcinoma-associated fibroblasts (CAF) may promote breast carcinogenesis and cancer progression. A better understanding of the changes that occur in fibroblasts during carcinogenesis and their influence on epithelial cell growth and behavior could lead to novel strategies for the prevention and treatment of breast cancer. To this end, the effect of CAF and normal breast-associated fibroblasts (NAF) on the growth of epithelial cells representative of pre-neoplastic breast disease was assessed. METHODS: NAF and CAF were grown with the nontumorigenic MCF10A epithelial cells and their more transformed, tumorigenic derivative, MCF10AT cells, in direct three-dimensional co-cultures on basement membrane material. The proliferation and apoptosis of MCF10A cells and MCF10AT cells were assessed by 5-bromo-2'-deoxyuridine labeling and TUNEL assay, respectively. Additionally, NAF and CAF were compared for expression of insulin-like growth factor II as a potential mediator of their effects on epithelial cell growth, by ELISA and by quantitative, real-time PCR. RESULTS: In relatively low numbers, both NAF and CAF suppressed proliferation of MCF10A cells. However, only NAF and not CAF significantly inhibited proliferation of the more transformed MCF10AT cells. The degree of growth inhibition varied among NAF or CAF from different individuals. In greater numbers, NAF and CAF have less inhibitory effect on epithelial cell growth. The rate of epithelial cell apoptosis was not affected by NAF or CAF. Mean insulin-like growth factor II levels were not significantly different in NAF versus CAF and did not correlate with the fibroblast effect on epithelial cell proliferation. CONCLUSION: Both NAF and CAF have the ability to inhibit the growth of pre-cancerous breast epithelial cells. NAF have greater inhibitory capacity than CAF, suggesting that the ability of fibroblasts to inhibit epithelial cell proliferation is lost during breast carcinogenesis. Furthermore, as the degree of transformation of the epithelial cells increased they became resistant to the growth-inhibitory effects of CAF. Insulin-like growth factor II could not be implicated as a contributor to this differential effect of NAF and CAF on epithelial cell growth.

O-GlcNAc modification is an endogenous inhibitor of the proteasome.

The ubiquitin proteasome system classically selects its substrates for degradation by tagging them with ubiquitin. Here, we describe another means of controlling proteasome function in a global manner. The 26S proteasome can be inhibited by modification with the enzyme, O-GlcNAc transferase (OGT). This reversible modification of the proteasome inhibits the proteolysis of the transcription factor Sp1 and a hydrophobic peptide through inhibition of the ATPase activity of 26S proteasomes. The Rpt2 ATPase in the mammalian proteasome 19S cap is modified by O-GlcNAc in vitro and in vivo and as its modification increases, proteasome function decreases. This mechanism may couple proteasomes to the general metabolic state of the cell. The O-GlcNAc modification of proteasomes may allow the organism to respond to its metabolic needs by controlling the availability of amino acids and regulatory proteins.

Suppression of Neu-induced mammary tumor growth in cyclin D1 deficient mice is compensated for by cyclin E.

Amplification and/or overexpression of the receptor tyrosine kinase HER2/Neu and the cell cycle regulatory gene cyclin D1 are frequently associated with human breast cancer. We studied the functional significance of cyclin D1 in Neu-induced mammary oncogenesis by developing mice overexpressing either wild-type or mutant Neu in a cyclin D1 deficient background. The absence of cyclin D1 suppresses mammary tumor formation induced by the wild-type or activated mutant form of Neu, which promote multi- and single-step progression of tumorigenesis, respectively. These data indicate that cyclin D1 is preferentially required for Neu-mediated signal transduction pathways in mammary oncogenesis. Significantly, 35% of mutant Neu/cyclin D1(-/-) mice regained mammary tumor potential due to compensation by cyclin E. Thus, shared targets of cyclins D1 and E are important in modulating Neu function in mammary tumorigenesis. Our results imply that the combinatorial inhibition of cyclins D1 and E might be useful in the treatment of malignancies induced by Neu.