A novel Hsp90 inhibitor AT13387 induces senescence in EBV-positive nasopharyngeal carcinoma cells and suppresses tumor formation

Background: Nasopharyngeal carcinoma (NPC) is an epithelial malignancy strongly associated with Epstein-Barr virus (EBV). AT13387 is a novel heat shock protein 90 (Hsp90) inhibitor, which inhibits the chaperone function of Hsp90 and reduces expression of Hsp90-dependent client oncoproteins. This study aimed to evaluate both the in vitro and in vivo antitumor effects of AT13387 in the EBV-positive NPC cell line C666-1.Results: Our results showed that AT13387 inhibited C666-1 cell growth and induced cellular senescence with the downregulation of multiple Hsp90 client oncoproteins EGFR, AKT, CDK4, and restored the protein expression of negative cell cycle regulator p27. We also studied the ability of AT13387 to restore p27 expression by downregulation of AKT and the p27 ubiquitin mediator, Skp2, using AKT inhibitor and Skp2 siRNA. In the functional study, AT13387 inhibited cell migration with downregulation of a cell migration regulator, HDAC6, and increased the acetylation and stabilization of α-tubulin. We also examined the effect of AT13387 on putative cancer stem cells (CSC) by 3-D tumor sphere formation assay. AT13387 effectively reduced both the number and size of C666-1 tumor spheres with decreased expression of NPC CSC-like markers CD44 and SOX2. In the in vivo study, AT13387 significantly suppressed tumor formation in C666-1 NPC xenografts.Conclusion: AT13387 suppressed cell growth, cell migration, tumor sphere formation and induced cellular senescence on EBV-positive NPC cell line C666-1. Also, the antitumor effect of AT13387 was demonstrated in an in vivo model. This study provided experimental evidence for the preclinical value of using AT13387 as an effective antitumor agent in treatment of NPC. © 2013 Chan et al.; licensee BioMed Central Ltd.published_or_final_versio

Modeling the heterogeneity in risk of progression to Alzheimer's disease across cognitive profiles in mild cognitive impairment

Heterogeneity in risk of conversion to Alzheimer's disease (AD) among individuals with mild cognitive impairment (MCI) is well known. Novel statistical methods that are based on partially ordered set (poset) models can be used to create models that provide detailed and accurate information about performance with specific cognitive functions. This approach allows for the study of direct links between specific cognitive functions and risk of conversion to AD from MCI. It also allows for further delineation of multi-domain amnestic MCI, in relation to specific non-amnestic cognitive deficits, and the modeling of a range of episodic memory functioning levels. From the Alzheimer's Disease Neuroimaging Initiative (ADNI) study, conversion at 24 months of 268 MCI subjects was analyzed. It was found that 101 of those subjects (37.7%) converted to AD within that time frame. Poset models were then used to classify cognitive performance for MCI subjects. Respective observed conversion rates to AD were calculated for various cognitive subgroups, and by APOE e4 allele status. These rates were then compared across subgroups. The observed conversion rate for MCI subjects with a relatively lower functioning with a high level of episodic memory at baseline was 61.2%. In MCI subjects who additionally also had relatively lower perceptual motor speed functioning and at least one APOE e4 allele, the conversion rate was 84.2%. In contrast, the observed conversion rate was 9.8% for MCI subjects with a relatively higher episodic memory functioning level and no APOE e4 allele. Relatively lower functioning with cognitive flexibility and perceptual motor speed by itself also appears to be associated with higher conversion rates. Among MCI subjects, specific baseline cognitive profiles that were derived through poset modeling methods, are clearly associated with differential rates of conversion to AD. More precise delineation of MCI by such cognitive functioning profiles, including notions such as multidomain amnestic MCI, can help in gaining further insight into how heterogeneity arises in outcomes. Poset-based modeling methods may be useful for providing more precise classification of cognitive subgroups among MCI for imaging and genetics studies, and for developing more efficient and focused cognitive test batteries

Clinical Positioning of the IAP Antagonist Tolinapant (ASTX660) in Colorectal Cancer

Inhibitors of apoptosis proteins (IAPs) are intracellular proteins, with important roles in regulating cell death, inflammation and immunity. Here, we examined the clinical and therapeutic relevance of IAPs in colorectal cancer (CRC). We found that elevated expression of cIAP1 and cIAP2 (but not XIAP) significantly correlated with poor prognosis in microsatellite stable (MSS) stage-III CRC patients treated with 5-Fluorouracil (5FU)-based adjuvant chemotherapy, suggesting their involvement in promoting chemo-resistance. A novel IAP antagonist tolinapant (ASTX660) potently and rapidly downregulated cIAP1 in CRC models, demonstrating its robust on-target efficacy. In cells co-cultured with TNFα to mimic an inflammatory tumor microenvironment, tolinapant induced caspase-8-dependent apoptosis in CRC cell line models; however, the extent of apoptosis was limited due to inhibition by the caspase-8 paralogs FLIP and, unexpectedly, caspase-10. Importantly, tolinapant-induced apoptosis was augmented by FOLFOX in human CRC and murine organoid models in vitro and in vivo, due (at least in part) to FOLFOX-induced downregulation of Class-I histone deacetylases, leading to acetylation of the FLIP-binding partner Ku70 and downregulation of FLIP. Moreover, the effects of FOLFOX could be phenocopied using the clinically-relevant Class-I HDAC inhibitor, entinostat, which also induced acetylation of Ku70 and FLIP downregulation. Further analyses revealed that caspase-8-knockout RIPK3-positive CRC models were sensitive to tolinapant-induced necroptosis, an effect that could be exploited in caspase-8-proficient models using the clinically relevant caspase inhibitor emricasan. Our study provides evidence for immediate clinical exploration of tolinapant in combination with FOLFOX in poor prognosis MSS CRC with elevated cIAP1/2 expression

Ubiquitylation of MLKL at lysine 219 positively regulates necroptosis-induced tissue injury and pathogen clearance

Necroptosis is a form of cell death characterized by membrane rupture via MLKL oligomerization, although mechanistic details remain unclear. Here, the authors show that MLKL ubiquitylation of K219 facilitates high-order membrane assembly and subsequent rupture, promoting cytotoxicity. Necroptosis is a lytic, inflammatory form of cell death that not only contributes to pathogen clearance but can also lead to disease pathogenesis. Necroptosis is triggered by RIPK3-mediated phosphorylation of MLKL, which is thought to initiate MLKL oligomerisation, membrane translocation and membrane rupture, although the precise mechanism is incompletely understood. Here, we show that K63-linked ubiquitin chains are attached to MLKL during necroptosis and that ubiquitylation of MLKL at K219 significantly contributes to the cytotoxic potential of phosphorylated MLKL. The K219R MLKL mutation protects animals from necroptosis-induced skin damage and renders cells resistant to pathogen-induced necroptosis. Mechanistically, we show that ubiquitylation of MLKL at K219 is required for higher-order assembly of MLKL at membranes, facilitating its rupture and necroptosis. We demonstrate that K219 ubiquitylation licenses MLKL activity to induce lytic cell death, suggesting that necroptotic clearance of pathogens as well as MLKL-dependent pathologies are influenced by the ubiquitin-signalling system

Cbx2, a polycomb group gene, is required for Sry gene expression in mice

Mice lacking the function of the polycomb group protein CBX2(chromobox homolog 2; also known as M33) show defects in gonadal, adrenal, and splenic development. In particular, XY knockout (KO) mice develop ovaries but not testes, and the gonads are hypoplastic in both sexes. However, how CBX2 regulates development of these tissues remains largely unknown. In the present study, we used microarray, RT-PCR, and immunohistochemical analyses to show that the expression of Sry, Sox9, Lhx9, Ad4BP/SF-1, Dax-1, Gata4, Arx, and Dmrt1, genes encoding transcription factors essential for gonadal development, is affected in Cbx2 KO gonads. Male-to-female sex reversal in Cbx2 KO mice was rescued by crossing them with transgenic mice displaying forced expression of Sry or Sox9. However, testes remained hypoplastic in these mice, indicating that the size and the sex of the gonad are determined by different sets of genes. Our study implicates Cbx2 in testis differentiation through regulating Sry gene expression. (Endocrinology 153: 913-924, 2012

Differential modulatory effects of Annexin 1 on nitric oxide synthase induction by lipopolysaccharide in macrophages

Annexin-1 (ANXA1) is a glucocorticoid-regulated protein that modulates the effects of bacterial lipopolysaccharide (LPS) on macrophages. Exogenous administration of peptides derived from the N-terminus of ANXA1 reduces LPS-stimulated inducible nitric oxide synthase (iNOS) expression, but the effects of altering the endogenous expression of this protein are unclear. We transfected RAW264.7 murine macrophage-like cell lines to over-express constitutively ANXA1 and investigated whether this protein modulates the induction of iNOS, cyclooxygenase-2 (COX-2) and tumour necrosis factor-α (TNF-α) in response to LPS. In contrast to exogenous administration of N-terminal peptides, endogenous over-expression of ANXA1 results in up-regulation of LPS-induced iNOS protein expression and activity. However, levels of iNOS mRNA are unchanged. ANXA1 has no effect on COX-2 or TNF-α production in response to LPS. In experiments to investigate the mechanisms underlying these phenomena we observed that activation of signalling proteins classically associated with iNOS transcription was unaffected. Over-expression of ANXA1 constitutively activates extracellular signal regulated kinase (ERK)-1 and ERK-2, components of a signalling pathway not previously recognized as regulating LPS-induced iNOS expression. Inhibition of ERK activity, by the inhibitor U0126, reduced LPS-induced iNOS expression in our cell lines. Over-expression of ANXA1 also modified LPS-induced phosphorylation of the ERK-regulated translational regulation factor eukaryotic initiation factor 4E. Our data suggest that ANXA1 may modify iNOS levels by post-transcriptional mechanisms. Thus differential effects on iNOS expression in macrophages are seen when comparing acute administration of ANXA1 peptides versus the chronic endogenous over-expression of ANXA1