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Breast cancer is the most common cancer type for women in the western world. Despite decades of research, the molecular processes associated with breast cancer progression are still inadequately defined. Here, we focus on the systematic alteration of metabolism by using the state of the art metabolomic profiling techniques to investigate the changes of 157 metabolites during the progression of normal mouse mammary epithehal cells to an isogenic series of mammary tumor cell lines with increasing metastatic potentials. Our results suggest a two-step metabolic progression hypothesis during the acquisition of tumorigenic and metastatic abilities. Metabohite changes accompanying tumor progression are identified in the intracellular and secreted forms in several pathways, including glycolysis, the tricarboxyhic acid cycle, the pentose phosphate pathway, fatty acid and nucleotide biosynthesis, and the GSH-dependent antioxidative pathway. These results suggest possible biomarkers of breast cancer progression as well as opportunities of interrupting tumor progression through the targeting of metabolic pathways. [ABSTRACT FROM AUTHOR]

Metastatic spread of cancer to distant vital organs, including lung and bone, is the overwhelming cause of breast cancer mortality and morbidity. Effective treatment of systemic metastasis relies on the identification and functional characterization of metastasis mediators to multiple organs. Overexpression of the chemokine (C-C motif) ligand 2 (CCL2) is frequently associated with advanced tumor stage and metastatic relapse in breast cancer. However, the functional mechanism of CCL2 in promoting organ-specific metastasis of breast cancer has not been rigorously investigated. Here, we used organ-specific metastatic sublines of the MDA-MB-231 human breast cancer cell line to demonstrate that overexpression of CCL2 promotes breast cancer metastasis to both lung and bone. Conversely, blocking CCL2 function with a neutralizing antibody reduced lung and bone metastases. The enhancement of lung and bone metastases by CCL2 was associated with increased macrophage infiltration and osteoclast differentiation, respectively. By performing functional assays with primary cells isolated from the wild type, CCL2 and CCR2 knock-out mice, we showed that tumor cellderived CCL2 depends on its receptor CCR2 (chemokine, CC motif, receptor 2) expressed on stromal cells to exert its function in promoting macrophage recruitment and osteoclast differentiation. Overall, these data demonstrated that CCL2-expressing breast tumor cells engage CCR2+ stromal cells of monocytic origin, including macrophages and preosteoclasts, to facilitate colonization in lung and bone. Therefore, CCL2 and CCR2 are promising therapeutic targets for simultaneously inhibiting lung and bone metastasis of breast cancer. [ABSTRACT FROM AUTHOR]

The glycoprotein (Gp) Ib-IX-V complex is essential for platelet-mediated hemostasis and thrombosis. The cytoplasmic domain of its largest polypeptide subunit GpIbα possesses a binding region for filamin A, which links GpIb-IX-V to the platelet cytoskeleton. There is evidence that filamin A binding to GpIbα directs the surface expression of GpIb-IX. To investigate the mechanism of this effect, we examined GpIbα biosynthesis in Chinese hamster ovary (CHO) cells stably co-expressing wild-type or mutant GpIbα with GpIbβ, GpIX with and without filamin A. We observed that surface GpIbα expression is enhanced in CHO cells co-expressing human filamin A. In comparison with cells expressing only GpIbα, GpIbβ, and GpIX (CHO-GpIbα/βIX), lysates from CHO-GpIbα/βIX + filamin A-expressing cells showed greater amounts of immature, incompletely O-glycosylated and fully mature GpIbα, but lesser amounts of the -∼15-kDa C-terminal peptide released when the extracellular domain of GpIbα is cleaved by proteases. When filamin A binding is eliminated by truncation of GpIbα at C-terminal residue 557 or by a deletion between amino acids 560–570, the decreased synthesis of mature GpIbα is accompanied by decreased immature GpIbα and by an increased immunodetectable C-terminal peptide. The synthesis of mature GpIbα in CHO-GpIbα/βIX cells is eliminated by brefeldin A (which inhibits transport out of the endoplasmic reticulum (ER)) and restored by lactacystin (which inhibits proteasomal degradation). These results suggest that GpIbα binds to filamin A within the ER and that filamin A binding directs post-ER trafficking of GpIbα to the cell surface. [ABSTRACT FROM AUTHOR]

Engineering a highly tumor microenvironment-responsive nanoplatform toward effective chemotherapy has always been a challenge in targeted cancer treatment. Metal-organic frameworks are a promising delivery system to reformulate previously approved drugs for enhanced chemotherapy, such as disulfiram (DSF). Herein, a tumor microenvironment-activated metal-organic framework--based nanoplatform DSF@MOF-199@FA has been fabricated to realize amplified oxidative stress--induced enhanced chemotherapy. Our results unveil that the copper ions and DSF released by DSF@MOF-199@FA in an acidic environment can be converted into toxic bis(N, Ndiethyl dithiocarbamate) copper and then induce cell apoptosis. Simultaneously, we determined that the apoptosis outcome is further promoted by amplified oxidative stress through effective generation of reactive oxygen species and GSH elimination. In conclusion, this work provides a promising platform for effective anticancer treatment. [ABSTRACT FROM AUTHOR]

The aspariginyl hydroxylase human factor inhibiting hypoxia-inducible factor (FIH) is an important regulator of the transcriptional activity of hypoxia-inducible factor. FIH also catalyzes the hydroxylation of asparaginyl and other residues in ankyrin repeat domain–containing proteins, including apoptosis stimulating of p53 protein (ASPP) family members. ASPP2 is reported to undergo a single FIH-catalyzed hydroxylation at Asn-986. We report biochemical and crystallographic evidence showing that FIH catalyzes the unprecedented posttranslational hydroxylation of both asparaginyl residues in “VNVN” and related motifs of ankyrin repeat domains in ASPPs (i.e., ASPP1, ASPP2, and iASPP) and the related ASB11 and p18-INK4C proteins. Our biochemical results extend the substrate scope of FIH catalysis and may have implications for its biological roles, including in the hypoxic response and ASPP family function. [ABSTRACT FROM AUTHOR]

Chronic inflammation may contribute to insulin resistance via molecular cross-talk between pathways for pro-inflammatory and insulin signaling. Interleukin 1 receptor-associated kinase 1 (IRAK-1) mediates pro-inflammatory signaling via IL-1 receptor/Toll-like receptors, which may contribute to insulin resistance, but this hypothesis is untested. Here, we used male Irak1 null (k/o) mice to investigate the metabolic role of IRAK-1. C57BL/6 wild-type (WT) and k/o mice had comparable body weights on low-fat and high-fat diets (LFD and HFD, respectively). After 12 weeks on LFD (but not HFD), k/o mice (versus WT) had substantially improved glucose tolerance (assessed by the intraperitoneal glucose tolerance test (IPGTT)). As assessed with the hyperinsulinemic euglycemic glucose clamp technique, insulin sensitivity was 30% higher in the Irak1 k/o mice on chow diet, but the Irak1 deletion did not affect IPGTT outcomes in mice on HFD, suggesting that the deletion did not overcome the impact of obesity on glucose tolerance. Moreover, insulin-stimulated glucose-disposal rates were higher in the k/o mice, but we detected no significant difference in hepatic glucose production rates (± insulin infusion). Positron emission/computed tomography scans indicated higher insulin-stimulated glucose uptake in muscle, but not liver, in Irak1 k/o mice in vivo. Moreover, insulin-stimulated phosphorylation of Akt was higher in muscle, but not in liver, from Irak1 k/o mice ex vivo. In conclusion, Irak1 deletion improved muscle insulin sensitivity, with the effect being most apparent in LFD mice. [ABSTRACT FROM AUTHOR]

Biochemical and cell-based studies have identified the G0S2 (G0/G1 switch gene 2) as a selective inhibitor of the key intracel-lular triacylglycerol hydrolase, adipose triglyceride lipase. To better understand the physiological role of G0S2, we constructed an adipose tissue-specific G0S2 transgenic mouse model. In comparison with wild type animals, the transgenic mice exhibited a significant increase in overall fat mass and a decrease in peripheral triglyceride accumulation. Basal and adrenergically stimulated lipolysis was attenuated in adipose explants isolated from the transgenic mice. Following fasting or injection of a β3-adrenergic agonist, in vivo lipolysis and keto-genesis were decreased in G0S2 transgenic mice when compared with wild type animals. Consequently, adipose overexpression of G0S2 prevented the "switch" of energy substrate from carbohydrates to fatty acids during fasting. Moreover, G0S2 overexpression promoted accumulation of more and larger lipid droplets in brown adipocytes without impacting either mitochondrial morphology or expression of oxidative genes. This phenotypic change was accompanied by defective cold adaptation. Furthermore, feeding with a high fat diet caused a greater gain of both body weight and adiposity in the transgenic mice. The transgenic mice also displayed a decrease in fasting plasma levels of free fatty acid, triglyceride, and insulin as well as improved glucose and insulin tolerance. Cumulatively, these results indicate that fat-specific G0S2 overexpression uncouples adiposity from insulin sensitivity and overall metabolic health through inhibiting adipose lipolysis and decreasing circulating fatty acids. [ABSTRACT FROM AUTHOR]

TNF-α potently stimulates basal lipolysis in adipocytes, which may contribute to hyperlipidemia and peripheral insulin resistance in obesity. Recent studies show that adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL) act sequentially in catalyzing the first two steps of adipose lipolysis in response to β-adrenergic stimulation. Here, we sought to determine their functional roles in TNF-α-induced lipolysis. Silencing of ATGL expression in adipocytes almost completely abolished basal and TNF-α-induced glycerol release. In comparison, the glycerol release under the same conditions was only partially decreased upon reduction in expression of either HSL or the ATGL coactivator CGI-58. Interestingly, overexpression of ATGL restored the lipolytic rates in cells with silenced HSL or CGI-58, indicating a predominant role for ATGL. While expression of ATGL, HSL and CGI-58 remains mostly unaffected, TNF-α treatment caused a rapid abrogation of the ATGL inhibitory protein G0S2. TNF-α drastically decreased the level of G0S2 mRNA, and the level of G0S2 protein could be maintained by inhibiting proteasomal protein degradation using MG-132. Furthermore, coexpression of G0S2 was able to significantly decrease TNF-α-stimulated lipolysis mediated by overexpressed ATGL or CGI-58. We propose that the early reduction in G0S2 content is permissive for TNF-α-induced lipolysis. [ABSTRACT FROM AUTHOR]