Aurora-A Mitotic Kinase Induces Endocrine Resistance through Down-Regulation of ERα Expression in Initially ERα+ Breast Cancer Cells

Development of endocrine resistance during tumor progression represents a major challenge in the management of estrogen receptor alpha (ERα) positive breast tumors and is an area under intense investigation. Although the underlying mechanisms are still poorly understood, many studies point towards the ‘cross-talk’ between ERα and MAPK signaling pathways as a key oncogenic axis responsible for the development of estrogen-independent growth of breast cancer cells that are initially ERα+ and hormone sensitive. In this study we employed a metastatic breast cancer xenograft model harboring constitutive activation of Raf-1 oncogenic signaling to investigate the mechanistic linkage between aberrant MAPK activity and development of endocrine resistance through abrogation of the ERα signaling axis. We demonstrate for the first time the causal role of the Aurora-A mitotic kinase in the development of endocrine resistance through activation of SMAD5 nuclear signaling and down-regulation of ERα expression in initially ERα+ breast cancer cells. This contribution is highly significant for the treatment of endocrine refractory breast carcinomas, because it may lead to the development of novel molecular therapies targeting the Aurora-A/SMAD5 oncogenic axis. We postulate such therapy to result in the selective eradication of endocrine resistant ERαlow/− cancer cells from the bulk tumor with consequent benefits for breast cancer patients

TGF-β Inducible Early Gene 1 Regulates Osteoclast Differentiation and Survival by Mediating the NFATc1, AKT, and MEK/ERK Signaling Pathways

TGF-β Inducible Early Gene-1 (TIEG1) is a Krüppel-like transcription factor (KLF10) that was originally cloned from human osteoblasts as an early response gene to TGF-β treatment. As reported previously, TIEG1−/− mice have decreased cortical bone thickness and vertebral bone volume and have increased spacing between the trabeculae in the femoral head relative to wildtype controls. Here, we have investigated the role of TIEG1 in osteoclasts to further determine their potential role in mediating this phenotype. We have found that TIEG1−/− osteoclast precursors differentiated more slowly compared to wildtype precursors in vitro and high RANKL doses are able to overcome this defect. We also discovered that TIEG1−/− precursors exhibit defective RANKL-induced phosphorylation and accumulation of NFATc1 and the NFATc1 target gene DC-STAMP. Higher RANKL concentrations reversed defective NFATc1 signaling and restored differentiation. After differentiation, wildtype osteoclasts underwent apoptosis more quickly than TIEG1−/− osteoclasts. We observed increased AKT and MEK/ERK signaling pathway activation in TIEG1−/− osteoclasts, consistent with the roles of these kinases in promoting osteoclast survival. Adenoviral delivery of TIEG1 (AdTIEG1) to TIEG1−/− cells reversed the RANKL-induced NFATc1 signaling defect in TIEG1−/− precursors and eliminated the differentiation and apoptosis defects. Suppression of TIEG1 with siRNA in wildtype cells reduced differentiation and NFATc1 activation. Together, these data provide evidence that TIEG1 controls osteoclast differentiation by reducing NFATc1 pathway activation and reduces osteoclast survival by suppressing AKT and MEK/ERK signaling

Two-dimensional NMR lineshape analysis

NMR titration experiments are a rich source of structural, mechanistic, thermodynamic and kinetic information on biomolecular interactions, which can be extracted through the quantitative analysis of resonance lineshapes. However, applications of such analyses are frequently limited by peak overlap inherent to complex biomolecular systems. Moreover, systematic errors may arise due to the analysis of two-dimensional data using theoretical frameworks developed for one-dimensional experiments. Here we introduce a more accurate and convenient method for the analysis of such data, based on the direct quantum mechanical simulation and fitting of entire two-dimensional experiments, which we implement in a new software tool, TITAN (TITration ANalysis). We expect the approach, which we demonstrate for a variety of protein-protein and protein-ligand interactions, to be particularly useful in providing information on multi-step or multi-component interactions

Promoter polymorphism -119C/G in MYG1 (C12orf10) gene is related to vitiligo susceptibility and Arg4Gln affects mitochondrial entrance of Myg1

<p>Abstract</p> <p>Background</p> <p><it>MYG1 </it>(<it>Melanocyte proliferating gene 1</it>, also C12orf10 in human) is a ubiquitous nucleo-mitochondrial protein, involved in early developmental processes and in adult stress/illness conditions. We recently showed that <it>MYG1 </it>mRNA expression is elevated in the skin of vitiligo patients. Our aim was to examine nine known polymorphisms in the <it>MYG1 </it>gene, to investigate their functionality, and to study their association with vitiligo susceptibility.</p> <p>Methods</p> <p>Nine single nucleotide polymorphisms (SNPs) in the <it>MYG1 </it>locus were investigated by SNPlex assay and/or sequencing in vitiligo patients (n = 124) and controls (n = 325). <it>MYG1 </it>expression in skin biopsies was detected by quantitative-real time PCR (Q-RT-PCR) and polymorphisms were further analysed using luciferase and YFP reporters in the cell culture.</p> <p>Results</p> <p>Control subjects with -119G promoter allele (rs1465073) exhibited significantly higher <it>MYG1 </it>mRNA levels than controls with -119C allele (<it>P </it>= 0.01). Higher activity of -119G promoter was confirmed by luciferase assay. Single marker association analysis showed that the -119G allele was more frequent in vitiligo patients (47.1%) compared to controls (39.3%, <it>P </it>< 0.05, OR 1.37, 95%CI 1.02-1.85). Analysis based on the stage of progression of the vitiligo revealed that the increased frequency of -119G allele occurred prevalently in the group of patients with active vitiligo (n = 86) compared to the control group (48.2% <it>versus </it>39.3%, <it>P </it>< 0.05; OR 1.44, 95%CI 1.02-2.03). Additionally, we showed that glutamine in the fourth position (in Arg4Gln polymorphism) completely eliminated mitochondrial entrance of YFP-tagged Myg1 protein in cell culture. The analysis of available EST, cDNA and genomic DNA sequences revealed that Myg1 4Gln allele is remarkably present in human populations but is never detected in homozygous state according to the HapMap database.</p> <p>Conclusions</p> <p>Our study demonstrated that both <it>MYG1 </it>promoter polymorphism -119C/G and Arg4Gln polymorphism in the mitochondrial signal of Myg1 have a functional impact on the regulation of the <it>MYG1 </it>gene and promoter polymorphism (-119C/G) is related with suspectibility for actively progressing vitiligo.</p

Insights into the Mechanism of Bovine CD38/NAD+Glycohydrolase from the X-Ray Structures of Its Michaelis Complex and Covalently-Trapped Intermediates

Bovine CD38/NAD+glycohydrolase (bCD38) catalyses the hydrolysis of NAD+ into nicotinamide and ADP-ribose and the formation of cyclic ADP-ribose (cADPR). We solved the crystal structures of the mono N-glycosylated forms of the ecto-domain of bCD38 or the catalytic residue mutant Glu218Gln in their apo state or bound to aFNAD or rFNAD, two 2′-fluorinated analogs of NAD+. Both compounds behave as mechanism-based inhibitors, allowing the trapping of a reaction intermediate covalently linked to Glu218. Compared to the non-covalent (Michaelis) complex, the ligands adopt a more folded conformation in the covalent complexes. Altogether these crystallographic snapshots along the reaction pathway reveal the drastic conformational rearrangements undergone by the ligand during catalysis with the repositioning of its adenine ring from a solvent-exposed position stacked against Trp168 to a more buried position stacked against Trp181. This adenine flipping between conserved tryptophans is a prerequisite for the proper positioning of the N1 of the adenine ring to perform the nucleophilic attack on the C1′ of the ribofuranoside ring ultimately yielding cADPR. In all structures, however, the adenine ring adopts the most thermodynamically favorable anti conformation, explaining why cyclization, which requires a syn conformation, remains a rare alternate event in the reactions catalyzed by bCD38 (cADPR represents only 1% of the reaction products). In the Michaelis complex, the substrate is bound in a constrained conformation; the enzyme uses this ground-state destabilization, in addition to a hydrophobic environment and desolvation of the nicotinamide-ribosyl bond, to destabilize the scissile bond leading to the formation of a ribooxocarbenium ion intermediate. The Glu218 side chain stabilizes this reaction intermediate and plays another important role during catalysis by polarizing the 2′-OH of the substrate NAD+. Based on our structural analysis and data on active site mutants, we propose a detailed analysis of the catalytic mechanism

Epigenetic polypharmacology: from combination therapy to multitargeted drugs

The modern drug discovery process has largely focused its attention in the so-called magic bullets, single chemical entities that exhibit high selectivity and potency for a particular target. This approach was based on the assumption that the deregulation of a protein was causally linked to a disease state, and the pharmacological intervention through inhibition of the deregulated target was able to restore normal cell function. However, the use of cocktails or multicomponent drugs to address several targets simultaneously is also popular to treat multifactorial diseases such as cancer and neurological disorders. We review the state of the art with such combinations that have an epigenetic target as one of their mechanisms of action. Epigenetic drug discovery is a rapidly advancing field, and drugs targeting epigenetic enzymes are in the clinic for the treatment of hematological cancers. Approved and experimental epigenetic drugs are undergoing clinical trials in combination with other therapeutic agents via fused or linked pharmacophores in order to benefit from synergistic effects of polypharmacology. In addition, ligands are being discovered which, as single chemical entities, are able to modulate multiple epigenetic targets simultaneously (multitarget epigenetic drugs). These multiple ligands should in principle have a lower risk of drug-drug interactions and drug resistance compared to cocktails or multicomponent drugs. This new generation may rival the so-called magic bullets in the treatment of diseases that arise as a consequence of the deregulation of multiple signaling pathways provided the challenge of optimization of the activities shown by the pharmacophores with the different targets is addressed

Comparison of tendon fiber structures between wild type and TIEG knockout mice using synchrotron microdiffraction

International audienc