Breast tumor cells with PI3K mutation or HER2 amplification are selectively addicted to Akt signaling

Background

Dysregulated PI3K/Akt signaling occurs commonly in breast cancers and is due to HER2 amplification, PI3K mutation or PTEN inactivation. The objective of this study was to determine the role of Akt activation in breast cancer as a function of mechanism of activation and whether inhibition of Akt signaling is a feasible approach to therapy.

Methodology/Principal Findings

A selective allosteric inhibitor of Akt kinase was used to interrogate a panel of breast cancer cell lines characterized for genetic lesions that activate PI3K/Akt signaling: HER2 amplification or PI3K or PTEN mutations in order to determine the biochemical and biologic consequences of inhibition of this pathway. A variety of molecular techniques and tissue culture and in vivo xenograft models revealed that tumors with mutational activation of Akt signaling were selectively dependent on the pathway. In sensitive cells, pathway inhibition resulted in D-cyclin loss, G1 arrest and induction of apoptosis, whereas cells without pathway activation were unaffected. Most importantly, the drug effectively inhibited Akt kinase and its downstream effectors in vivo and caused complete suppression of the growth of breast cancer xenografts with PI3K mutation or HER2 amplification, including models of the latter selected for resistance to Herceptin. Furthermore, chronic administration of the drug was well-tolerated, causing only transient hyperglycemia without gross toxicity to the host despite the pleiotropic normal functions of Akt.

Conclusions/Significance

These data demonstrate that breast cancers with PI3K mutation or HER2 amplification are selectively dependent on Akt signaling, and that effective inhibition of Akt in tumors is feasible and effective in vivo. These findings suggest that direct inhibition of Akt may represent a therapeutic strategy for breast and other cancers that are addicted to the pathway including tumors with resistant to Herceptin.     

Covalent attachment of proteins to solid supports and surfaces via Sortase-mediated ligation

Background

There is growing interest in the attachment of proteins to solid supports for the development of supported catalysts, affinity matrices, and micro devices as well as for the development of planar and bead based protein arrays for multiplexed assays of protein concentration, interactions, and activity. A critical requirement for these applications is the generation of a stable linkage between the solid support and the immobilized, but still functional, protein.

Methodology

Solid supports including crosslinked polymer beads, beaded agarose, and planar glass surfaces, were modified to present an oligoglycine motif to solution. A range of proteins were ligated to the various surfaces using the Sortase A enzyme of S. aureus. Reactions were carried out in aqueous buffer conditions at room temperature for times between one and twelve hours.

Conclusions

The Sortase A transpeptidase of S. aureus provides a general, robust, and gentle approach to the selective covalent immobilization of proteins on three very different solid supports. The proteins remain functional and accessible to solution. Sortase mediated ligation is therefore a straightforward methodology for the preparation of solid supported enzymes and bead based assays, as well as the modification of planar surfaces for microanalytical devices and protein arrays.     

Cutaneous and Mucosal Phaeohyphomycosis Caused by Exophiala spinifera in a Pregnant Patient: Case Report and Literature Review

We report a case of mucocutaneous phaeohyphomycosis caused by Exophiala spinifera. Crusty plaques and nodules were major clinical features. Histological examination revealed brown yeast-like cells and hyphae. Mycological and molecular data identified E. spinifera as etiologic agent. Oral itraconazole was effective, which was in accordance with the results of in vitro susceptibility testing. We speculated that her pregnancy may play a role of risk factor in the infection by E. spinifera.     

TBC1D25 alleviates nonalcoholic steatohepatitis by inhibiting abnormal lipid accumulation and inflammation

Nonalcoholic fatty liver disease (NAFLD) is a strong stimulant of cardiovascular diseases, affecting one-quarter of the world's population. TBC1 domain family member 25 (TBC1D25) regulates the development of myocardial hypertrophy and cerebral ischemia–reperfusion injury; however, its effect on NAFLD/nonalcoholic steatohepatitis (NASH) has not been reported. In this study, we demonstrated that TBC1D25 expression is upregulated in NASH. TBC1D25 deficiency aggravated hepatic steatosis, inflammation, and fibrosis in NASH. In vitro tests revealed that TBC1D25 overexpression restrained NASH responses. Subsequent mechanistic validation experiments demonstrated that TBC1D25 interfered with NASH progression by inhibiting abnormal lipid accumulation and inflammation. TBC1D25 deficiency significantly promoted NASH occurrence and development. Therefore, TBC1D25 may potentially be used as a clinical therapeutic target for NASH treatment.     

Water-protein interplay reveals the specificity of α-lytic protease

Wild type and mutant α-lytic protease, differing by only one amino acid, have distinct specificities. Previous studies have shown that motion patterns of the binding pocket play an important role. However, it is still unclear how these differences are generated from a single amino acid mutation. Based on comparative molecular dynamics simulations using explicit and implicit solvent models, we studied the dynamic properties of both protein and water. The explicit solvent simulations showed specificity related differences in the energy landscapes and the power spectra between the two enzymes, whereas implicit solvent simulations did not. Moreover, the explicit solvent simulations demonstrated obvious distinctions in dynamic behaviors of water, such as their residence behaviors and hydrogen bonding. These results suggest that the interplay between water and enzyme is essential in determining the substrate specificity, and the detail knowledge of such interplay can greatly improve our understanding of bio-molecules.     

Characterization of the agent of "high plains disease": mass spectrometry determines the sequence of the disease-specific protein

The "32-kDa" protein specifically associated with high plains disease was characterized by time-of-flight mass spectrometry, after the agent had been isolated in pure culture by "vascular puncture inoculation," a novel mechanical means of transmission. Two isolates from different geographic locations each consisted of a mixture of subpopulations that were highly homologous to an amino acid sequence derived from a nucleotide sequence (U60141) deposited in GenBank trade mark by the Nebraska group as "the probable N-protein of high plains virus." However, the U60141 sequence was found to be incomplete; de novo sequencing of peptides produced by proteolytic digestions of the 32-kDa band from an SDS-PAGE separation showed that an additional 18 amino acid residues were present at the N terminus. BLAST (basic local alignment search tool) examination of the sequence showed no significant homology with any protein in the databases, indicating that the infectious agent of high plains disease is likely a member of a hitherto unclassified virus group.     

Keratinocyte growth factor‐2 intratracheal instillation significantly attenuates ventilator‐induced lung injury in rats

Preservation or restoration of normal alveolar epithelial barrier function is crucial for pulmonary oedema resolution. Keratinocyte growth factor‐2 (KGF‐2), a potent epithelial cell mitogen, may have a role in preventing ventilator‐induced lung injury (VILI), which occurs frequently in mechanically ventilated patients. The aim of the study was to test the role of KGF‐2 in VILI in rats. Forty healthy adult male Sprague‐Dawley rats were randomly allocated into four groups, where rats in Groups HVZP (high‐volume zero positive end‐expiratory pressure) and HVZP+KGF‐2 were given intratracheally equal PBS and 5 mg/kg KGF‐2 72 hrs before 4 hrs HVZP ventilation (20 ml/kg), respectively, while PBS and KGF‐2 were administered in the same manner in Groups Control and KGF‐2, which underwent tracheotomy only with spontaneous breathing. Inflammatory cytokines (tumour necrosis factor‐α, macrophage inflammatory protein 2), neutrophil and total protein levels in bronchoalveolar lavage fluid and surfactant protein mRNA expression in lung tissue were detected; the number of alveolar type II cells, lung water content and lung morphology were also evaluated. The results indicate that pre‐treatment with KGF‐2 showed dramatic improvement in lung oedema and inflammation compared with HVZP alone, together with increased surfactant protein mRNA and alveolar type II cells. Our results suggest that KGF‐2 might be considered a promising prevention for human VILI or other acute lung injury diseases.     

Sarsaparilla (Smilax Glabra Rhizome) Extract Inhibits Migration and Invasion of Cancer Cells by Suppressing TGF-β1 Pathway

Sarsaparilla, also known as Smilax Glabra Rhizome (SGR), was shown to modulate immunity, protect against liver injury, lower blood glucose and suppress cancer. However, its effects on cancer cell adhesion, migration and invasion were unclear. In the present study, we found that the supernatant of water-soluble extract from SGR (SW) could promote adhesion, inhibit migration and invasion of HepG2, MDA-MB-231 and T24 cells in vitro, as well as suppress metastasis of MDA-MB-231 cells in vivo. Results of F-actin and vinculin dual staining showed the enhanced focal adhesion in SW-treated cells. Microarray analysis indicated a repression of TGF-β1 signaling by SW treatment, which was verified by real-time RT-PCR of TGF-β1-related genes and immunoblotting of TGFBR1 protein. SW was also shown to antagonize TGF-β1-promoted cell migration. Collectively, our study revealed a new antitumor function of Sarsaparilla in counteracting invasiveness of a subset of cancer cells by inhibiting TGF-β1 signaling.