One of the basic functions of insulin in the body is to inhibit lipolysis in adipocytes. Recently, we have found that insulin inhibits lipolysis and promotes triglyceride storage by decreasing transcription of adipose triglyceride lipase via the mTORC1-mediated pathway (P. Chakrabarti et al., Diabetes 59:775-781, 2010), although the mechanism of this effect remained unknown. Here, we used a genetic screen in Saccharomyces cerevisiae in order to identify a transcription factor that mediates the effect of Tor1 on the expression of the ATGL ortholog in yeast. This factor, Msn4p, has homologues in mammalian cells that form a family of early growth response transcription factors. One member of the family, Egr1, is induced by insulin and nutrients and directly inhibits activity of the ATGL promoter in vitro and expression of ATGL in cultured adipocytes. Feeding animals a high-fat diet increases the activity of mTORC1 and the expression of Egr1 while decreasing ATGL levels in epididymal fat. We suggest that the evolutionarily conserved mTORC1-Egr1-ATGL regulatory pathway represents an important component of the antilipolytic effect of insulin in the mammalian organism. C urrent epidemics of metabolic diseases, such as type 2 diabetes, cardiac dysfunction, hypertension, hepatic steatosis, etc., are largely caused by widespread obesity. Although obesity can affect human health via several different mechanisms, the bestestablished connection between obesity and metabolic disease is elevated and/or dysregulated levels of circulating free fatty acids (FFA). In addition to their direct pathological effects, superfluous FFA accumulate in the form of lipids, and their metabolic products in nonadipose peripheral tissues, such as liver, skeletal muscle, heart, and pancreas and cause detrimental effects on human health via mechanisms that are currently under intense investigation (1-5).The levels of circulating FFA depend primarily on the rates of lipolysis in the adipose tissue. One of the key physiological functions of insulin as the major anabolic hormone in the body is to restrain lipolysis and to promote fat storage in adipose tissue in the postprandial state. The failure of insulin to suppress lipolysis in adipocytes has been long considered as a very serious metabolic defect and one of the most important if not the most important causative factor of insulin resistance and diabetes mellitus (6, 7).Complete hydrolysis of triglycerides to glycerol and fatty acids is performed jointly by tri-, di-, and monoacylglyceride lipases (8-11). The recently discovered enzyme, adipose triglyceride lipase (ATGL; also known as desnutrin, PNPLA2, TTS2.2, and iPLA 2 ) (12-14), is responsible for the bulk of triacylglycerol hydrolase activity in various cells and represents the rate-limiting lipolytic enzyme. In every experimental model tested thus far, elevated ATGL expression increases, while attenuated ATGL expression decreases, both basal and cAMP-stimulated lipolysis (12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22). At the same time, ATGL h...

The transcription factor Egr-1 regulates the expression of numerous genes involved in differentiation, growth, and in response to environmental signals. Egr-1 activity is modulated in part through the binding of corepressors Nab1 and Nab2. Nab2 appears crucial for controlling Egr-1-mediated transactivation because it is a delayed early response gene, induced by the same stimuli that induce the immediate early gene Egr-1. To identify important elements regulating Nab2 expression, we cloned the human Nab2 gene and investigated the 5-region. The TATA-and initiator-less Nab2 promoter, located from ؊679 to ؊74 bp, contains a total of 11 Egr binding sites, including a cluster of multiple overlapping Egr/Sp1 sites between ؊329 and ؊260 bp. This region is critical for basal promoter activity as well as for maximum induction by phorbol esters. Electromobility shifts show that Sp1 binds to this region in normal and stimulated cells, whereas stimulation induces binding of Egr-1. In addition Egr-1 activates the Nab2 promoter in a pattern similar to phorbol esters, suggesting that Egr-1 is a major inducer of protein kinase C-mediated Nab2 induction. Depletion of Egr-1 by each of two distinct Egr-1 short-interfering RNAs reduces Nab2 expression and inducibility, confirming that Egr-1 is an important regulator of Nab2 expression. Transfection experiments show that Egr-1-induced Nab2 promoter activity is itself repressed by Nab2. These results indicate that Egr-1 mediates the induction of its own repressor, thereby preventing a permanent transactivation of Egr-1 target genes and a damaging overreaction in response to environmental signals.The zinc finger transcription factor early growth response (Egr 5 )-1 is an immediate early response gene that couples extracellular signals to the induction of cellular programs of differentiation, growth, and cell death through changes in the expression of Egr-1 target genes. Although Egr-1 expression is low or undetectable in resting cells, it is rapidly and transiently induced by a wide variety of environmental signals including growth factors, cytokines, and toxic substances (1). Egr-1 in turn activates the transcription of numerous target genes including growth factors that themselves induce Egr-1, as e.g. platelet-derived growth factor (PDGF), thereby establishing a positive autocrine feedback loop. Egr-1 activity is tightly regulated, in part through the function of two proteins, NGFI-A-binding protein (Nab)1 and Nab2, which prevents the permanent activation of Egr-1 target genes and signaling pathways such as those for PDGF (2), which could lead to cellular transformation. Nab proteins were first identified as Egr-1-binding proteins in a two-hybrid assay (3, 4). Nab2, which is also known as mader (melanoma-associated delayed early response gene), was independently identified as a nuclear protein overexpressed in human malignant melanoma (5). Binding of Nab1 and Nab2 to Egr-1 through interaction between the NCD1 (Nab conserved domain 1) and the R1 domain of Egr-1 has been shown to inhi...

The inducible zinc finger transcription factors EGR1, EGR2, and EGR3 regulate the expression of numerous genes involved in differentiation, growth, and response to extracellular signals. Their activity is modulated in part through NAB2 which is induced by the same stimuli. In melanoma and carcinoma cells EGR1 activates NAB2 expression. In T lymphocytes EGR2 and EGR3 have been shown to inhibit NAB2 expression. Therefore, we investigated the influence of EGR2 and EGR3 on NAB2 expression in melanoma and carcinoma cells. Here we show that like EGR1, EGR2 and EGR3 induce NAB2 expression in these cells. EGR1 and EGR3 act in concert on the NAB2 promoter and are more potent activators of NAB2 transcription than EGR2. EGR1-, EGR2-, and EGR3-induced NAB2 promoter activity is mediated through similar cis-regulatory elements and the activation by each EGR is repressed by NAB2. Kinetic studies suggest that induction of EGR1 leads to low NAB2 expression while EGR2 and EGR3 are necessary for maximal and sustained expression. As aleady shown for EGR1, reduction of EGR2 or EGR3 expression by siRNAs reduced endogenous NAB2 levels. Depletion of EGR3 also resulted in a reduction of EGR2 levels confirming EGR2 as a target gene of EGR3. Our results suggest that in many cells of neuroectodermal and epithelial origin EGR1, EGR2, and EGR3 activate NAB2 transcription which is in turn is repressed by NAB2, thus establishing a negative feedback loop. This points to a complex relationship between the EGR factors and NAB2 expression likely depending on the cellular context. KeywordsNAB2; early growth response; EGR; transcription factor; negative feedback loop; autoregulation; promoter The zinc finger transcription factors of the early growth response family (EGR 3 ) are immediate early response genes that are important for the induction of cellular programs of differentiation, proliferation, and cell death in response to environmental stimuli (Carter et al., 2007;Thiel et al., 2002;Unoki et al., 2003). The most prominent members of the EGR * Correspondence to: Judith P. Johnson, Institute for Immunology, Goethestrasse 31, Munich 80336, Germany. Tel.: +49-89-2180-75-660; Fax: +49-89-5160-2236; johnson@med.uni-muenchen.de. 3 The abbreviations used are: EGR, early growth response; NGFI-A, nerve growth factor-induced protein A; Nab, NGFI-A binding protein; MADER, melanoma associated delayed early response gene; SP1, specificity protein 1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; G6PD, glucose-6-phosphate dehydrogenase; AP2, activator protein 2; CREB, cAMP-responsive element binding protein; PDGF, platelet-derived growth factor; bFGF, basic fibroblast growth factor; VEGF, vascular endothelial growth factor; TCR, T cell receptor; siRNA, short-interfering RNA; PMA, phorbol myristate acetate; EMSA, electrophoretic mobility shift assay; PHLDA1, pleckstrin homology-like domain A-1; EGF, epidermal growth factor. NIH Public AccessAuthor Manuscript J Cell Biochem. Author manuscript; available in PMC 2011 September 1. NIH-PA Author Manu...

Background:The CD2AP adaptor facilitates cell signaling using its in-built interaction modules (SH3 domains). Results: RIN3 was characterized as a novel CD2AP SH3 binding protein by biophysical and biochemical methods. Conclusion: CD2AP has many potential interactors and is probably a cellular hub. Significance: We reveal how protein modules can interact with families of related recognition motifs rather than a single motif.

Elevated levels of p130(Cas)/BCAR1 (Crk-associated substrate/breast cancer antiestrogen resistance 1) are found in aggressive breast tumors and are associated with tamoxifen resistance of mammary cancers. p130(Cas) promotes the integration of protein complexes involved in multiple signaling pathways frequently deregulated in breast cancer. To elucidate mechanisms leading to p130(Cas) up-regulation in mammary carcinomas and during acquired tamoxifen resistance, the regulation of p130(Cas)/BCAR1 was studied. Because multiple putative binding motifs for the inducible transcription factor EGR1 were identified in the 5' region of BCAR1, the p130(Cas)/BCAR1 regulation by EGR1 and its coregulator NAB2 was investigated. Overexpression or short interfering RNA (siRNA)-mediated down-regulation of EGR1 or NAB2, and chromatin immunoprecipitations indicated that EGR1 and NAB2 act in concert to positively regulate p130(Cas)/BCAR1 expression in breast cancer cells. p130(Cas) depletion using siRNA showed that, in tamoxifen-sensitive MCF-7 cells, p130(Cas) regulates EGR1 and NAB2 expression, whereas in the derivative tamoxifen-resistant TAM-R cells, only NAB2 levels were influenced. BCAR1 messenger RNA and p130(Cas) protein were upregulated by phorbol esters following the kinetics of late response genes in MCF-7 but not in TAM-R cells. Thus, in MCF-7 cells, we identified a positive feedback loop where p130(Cas) positively regulates EGR1 and NAB2, which in turn induce p130(Cas) expression. Importantly, compared with MCF-7, enhanced NAB2 expression and increased EGR1 binding to the BCAR1 5' region observed in TAM-R may lead to the constitutively increased p130(Cas)/BCAR1 levels in TAM-R cells. The uncovered differences in this EGR1/NAB2/p130(Cas) network in MCF-7 versus TAM-R cells may also contribute to p130(Cas) up-regulation during acquired tamoxifen resistance.

Elevated expression of p130Cas (Crk-associated substrate)/BCAR1 (breast cancer antiestrogen resistance 1) in human breast tumors is a marker of poor prognosis and poor overall survival. p130Cas is a downstream target of the tyrosine kinase c-Src. Signaling mediated by p130Cas through its phosphorylated substrate domain (SD) and interaction with effector molecules directly promotes tumor progression. We previously developed a constitutively phosphorylated p130Cas SD molecule, Src*/SD (formerly referred to as Src*/CasSD), which acts as decoy molecule and attenuates the transformed phenotype in v-crk-transformed murine fibroblasts and human breast cancer cells. To test the function of this molecule in vivo, we established mouse mammary tumor virus (MMTV)-long terminal repeat-Src*/SD transgenic mice in which mammary gland development and tumor formation were analyzed. Transgenic expression of the Src*/SD molecule under the MMTV-long terminal repeat promoter did not interfere with normal mammary gland development or induce tumors in mice observed for up to 11 months. To evaluate the effects of the Src*/SD molecule on tumor development in vivo, we utilized the MMTV-polyoma middle T-antigen (PyMT) murine breast cancer model that depends on c-Src. PyMT mice crossed with Src*/SD mice displayed accelerated tumor formation. The earlier onset of tumors can be explained by the interaction of the Src* domain with PyMT and targeting the fused phosphorylated SD to the membrane. At membrane compartments, it might integrate membrane-associated active signaling complexes leading to increased proliferation measured by phospho-Histone H3 staining. Although these results were unexpected, they emphasize the importance of preventing the membrane association of Src*/SD when employed as decoy molecule.

Purpose Comprehensive genomic profiling identifying actionable molecular alterations aims to enable personalized treatment for cancer patients. The purpose of this analysis was to retrospectively assess the impact of personalized recommendations made by a multidisciplinary tumor board (MTB) on the outcome of patients with breast or gynecological cancers, who had progressed under standard treatment. Here, first experiences of our Comprehensive Cancer Center Molecular Tumor Board are reported. Methods All patients were part of a prospective local registry. 95 patients diagnosed with metastatic breast cancer or gynecological malignancies underwent extended molecular profiling. From May 2017 through March 2019, the MTB reviewed all clinical cases considering tumor profile and evaluated molecular alterations regarding further diagnostic and therapeutic recommendations. Results 95 patients with metastatic breast or gynecological cancers were discussed in the MTB (68% breast cancer, 20% ovarian cancer, 5% cervical cancer, 3% endometrial cancer and 4% others). Genes with highest mutation rate were PIK3CA and ERBB2. Overall, 34 patients (36%) received a biomarker-based targeted therapy recommendation. Therapeutic recommendations were implemented in nine cases; four patients experienced clinical benefit with a partial response or disease stabilization lasting over 4 months. Conclusion In the setting of a multidisciplinary molecular tumor board, a small but clinically meaningful group of breast and gynecological cancer patients benefits from comprehensive genomic profiling. Broad and successful implementation of precision medicine is complicated by patient referral at late stage disease and limited access to targeted agents and early clinical trials. Trial registration number 284-10 (03.05.2018).

Mutations in the hotspot regions of exon 9 and 13 of the POLE gene are very rare events in advanced pancreatic cancer. Thus, it is unlikely that POLE gene mutations contribute to genetic instability in the vast majority of aPDAC. POLE mutation does not serve as a relevant biomarker and should not be tested on a regular basis in PDAC.