Near universal detection of alterations in CTNNB1 and Wnt pathway regulators in desmoid-type fibromatosis by whole-exome sequencing and genomic analysis.

CTNNB1 mutations or APC abnormalities have been observed in ∼85% of desmoids examined by Sanger sequencing and are associated with Wnt/ß-catenin activation. We sought to identify molecular aberrations in "wild-type" tumors (those without CTNNB1 or APC alteration) and to determine their prognostic relevance. CTNNB1 was examined by Sanger sequencing in 117 desmoids; a mutation was observed in 101 (86%) and 16 were wild type. Wild-type status did not associate with tumor recurrence. Moreover, in unsupervised clustering based on U133A-derived gene expression profiles, wild-type and mutated tumors clustered together. Whole-exome sequencing of eight of the wild-type desmoids revealed that three had a CTNNB1 mutation that had been undetected by Sanger sequencing. The mutation was found in a mean 16% of reads (vs. 37% for mutations identified by Sanger). Of the other five wild-type tumors sequenced, two had APC loss, two had chromosome 6 loss, and one had mutation of BMI1. The finding of low-frequency CTNNB1 mutation or APC loss in wild-type desmoids was validated in the remaining eight wild-type desmoids; directed miSeq identified low-frequency CTNNB1 mutation in four and comparative genomic hybridization identified APC loss in one. These results demonstrate that mutations affecting CTNNB1 or APC occur more frequently in desmoids than previously recognized (111 of 117; 95%), and designation of wild-type genotype is largely determined by sensitivity of detection methods. Even true CTNNB1 wild-type tumors (determined by next-generation sequencing) may have genomic alterations associated with Wnt activation (chromosome 6 loss/BMI1 mutation), supporting Wnt/ß-catenin activation as the common pathway governing desmoid initiation.

Inhibition of human arginase I by substrate and product analogues.

Human arginase I is a binuclear manganese metalloenzyme that catalyzes the hydrolysis of L-arginine to generate L-ornithine and urea. We demonstrate that N-hydroxy-L-arginine (NOHA) binds to this enzyme with K(d)=3.6 microM, and nor-N-hydroxy-L-arginine (nor-NOHA) binds with K(d)=517 nM (surface plasmon resonance) or K(d) approximately 50 nM (isothermal titration calorimetry). Crystals of human arginase I complexed with NOHA and nor-NOHA afford 2.04 and 1.55 A resolution structures, respectively, which are significantly improved in comparison with previously-determined structures of the corresponding complexes with rat arginase I. Higher resolution structures clarify the binding interactions of the inhibitors. Finally, the crystal structure of the complex with L-lysine (K(d)=13 microM) is reported at 1.90 A resolution. This structure confirms the importance of hydrogen bond interactions with inhibitor alpha-carboxylate and alpha-amino groups as key specificity determinants of amino acid recognition in the arginase active site.

The secretion of IL-6 by CpG-ODN-treated cancer cells promotes T-cell immune responses partly through the TLR-9/AP-1 pathway in oral squamous cell carcinoma.

Increasing evidence suggests that communication between tumor and immune cells can alter the tumor microenvironment in ways that promote tumor development. The purpose of this study was to characterize the immune response elicited by TLR-9-activated OSCC cells, to identify the cytokines involved in the signaling pathway and to elucidate the molecular mechanism of this pathway in OSCC cells. MTS, flow cytometry and ELISA assay were used to evaluate T-cell immune responses, cancer cell proliferation and pro-inflammatory cytokine secretion, respectively. Western blot analysis, EMSA and ChIP assay were employed to detect the activity of the NF-κB and AP-1 signaling pathways. A marked response was observed when T-cells were co-cultured with supernatants from CpG-ODN-treated OSCC cells. This response was characterized by increased CD4+ and CD8+ T-cell proliferation and an increase in IFN-γ production by the CD4+ T-cell population. Treatment of OSCC cells with CpG-ODN resulted in an increase in IL-6 secretion as well as an increase in AP-1 binding activity to the IL-6 promoter. Moreover, blockage of the TLR-9/AP-1 pathway significantly decreased IL-6 expression and T-cell immune response. In human OSCC, the TLR-9 pathway, when stimulated by CpG-ODNs, promotes a T-cell immune response mediated by AP-1-activated IL-6 secretion. Although the complete molecular mechanism has yet to be understood, these findings provide evidence linking tumor cell activities to immune system responses. In addition, the TLR-9/AP-1/IL-6 pathway provides new therapeutic targets for the prevention and treatment of OSCC.

Binding of α,α-disubstituted amino acids to arginase suggests new avenues for inhibitor design.

Arginase is a binuclear manganese metalloenzyme that hydrolyzes L-arginine to form L-ornithine and urea, and aberrant arginase activity is implicated in various diseases such as erectile dysfunction, asthma, atherosclerosis, and cerebral malaria. Accordingly, arginase inhibitors may be therapeutically useful. Continuing our efforts to expand the chemical space of arginase inhibitor design and inspired by the binding of 2-(difluoromethyl)-L-ornithine to human arginase I, we now report the first study of the binding of α,α-disubstituted amino acids to arginase. Specifically, we report the design, synthesis, and assay of racemic 2-amino-6-borono-2-methylhexanoic acid and racemic 2-amino-6-borono-2-(difluoromethyl)hexanoic acid. X-ray crystal structures of human arginase I and Plasmodium falciparum arginase complexed with these inhibitors reveal the exclusive binding of the L-stereoisomer; the additional α-substituent of each inhibitor is readily accommodated and makes new intermolecular interactions in the outer active site of each enzyme. Therefore, this work highlights a new region of the protein surface that can be targeted for additional affinity interactions, as well as the first comparative structural insights on inhibitor discrimination between a human and a parasitic arginase.