Necroptosis is a caspase-independent form of regulated cell death that has been implicated in the development of a range of inflammatory, autoimmune and neurodegenerative diseases. The pseudokinase, Mixed Lineage Kinase Domain-Like (MLKL), is the most terminal known obligatory effector in the necroptosis pathway, and is activated following phosphorylation by Receptor Interacting Protein Kinase-3 (RIPK3). Activated MLKL translocates to membranes, leading to membrane destabilisation and subsequent cell death. However, the molecular interactions governing the processes downstream of RIPK3 activation remain poorly defined. Using a phenotypic screen, we identified seven heat-shock protein 90 (HSP90) inhibitors that inhibited necroptosis in both wild-type fibroblasts and fibroblasts expressing an activated mutant of MLKL. We observed a modest reduction in MLKL protein levels in human and murine cells following HSP90 inhibition, which was only apparent after 15 h of treatment. The delayed reduction in MLKL protein abundance was unlikely to completely account for defective necroptosis, and, consistent with this, we also found inhibition of HSP90 blocked membrane translocation of activated MLKL. Together, these findings implicate HSP90 as a modulator of necroptosis at the level of MLKL, a function that complements HSP90's previously demonstrated modulation of the upstream necroptosis effector kinases, RIPK1 and RIPK3.

The NAD+-dependent deacetylase, sirtuin 1 (SIRT1), has been recently been suspected to have a role in tumorigenesis. We investigated the expression of SIRT1 in pancreatic cancer and the effect of SIRT1-targeted RNA interference (RNAi) on cell proliferation and tumor formation in a pancreatic cancer cell line, PANC1. The expression of SIRT1 was investigated in 49 specimens of pancreatic cancer and adjacent normal pancreatic tissues. SIRT1 was overexpressed in pancreatic cancer tissues at both the mRNA and protein levels, with increased SIRT1 positivity associated with tumors from patients over 60 years old, tumors larger than 4 cm, higher TNM (extent of tumor (T), the extent of spread to lymph nodes (N), and presence of distant metastasis (M)) stage or the presence of lymph node or hepatic metastases. The PANC-1 was stably transfected with a SIRT1 small hairpin RNA (shRNA) expression plasmid and compared with untransfected and PANC-1-negative RNAi cells. Proliferation of PANC-1-SIRT1-RNAi cells was significantly reduced, accompanied by increased rates of apoptosis, G1 arrest and senescence. Furthermore, FOXO3a expression was markedly upregulated in PANC-1-SIRT1-RNAi cells, but no significant difference in p53 expression was observed. The invasive ability of PANC-1-SIRT1-RNAi cells was markedly reduced in vitro, which was linked to increased E-cadherin and reduced-MMP expression. Additionally, PANC-1-SIRT1-RNAi cells had a significantly reduced capacity to form tumors in vivo compared with untransfected and PANC-1-negative RNAi cells. These results suggest that SIRT1 may promote cell proliferation and tumor formation in pancreatic cancer, and downregulation of SIRT1 using shRNA could provide a novel therapeutic treatment. Gene Therapy (2011) 18, 920-928; doi:10.1038/gt.2011.81; published online 16 June 2011Keywords: SIRT1; RNAi; pancreatic cancer; proliferation; apoptosis INTRODUCTION Pancreatic cancer is one of the most aggressive solid malignancies, the third most common gastrointestinal malignancy, the fifth leading cause of cancer mortality and is a leading cause of cancer-related deaths in the developed world. 1 One key characteristic of pancreatic cancer is early systemic metastasis and extremely rapid local tumor progression. 2 Resistance to existing chemotherapeutic agents occurs commonly in pancreatic cancer therapy, 3 and therefore, there is an urgent need to elucidate the molecular mechanisms that underlie the malignant behavior of pancreatic cancer.It has been recently suspected that sirtuin 1 (SIRT1) is involved in tumorigenesis. 4,5 SIRT1 is one of seven members of the sirtuin family of nicotinamide adenine dinucleotide (NAD+)-dependent class III histone deacetylases, with sequence homology to the enzymatic domain of yeast silent information regulator 2 (sir2). 6,7 SIRT1 has a large number of histone and non-histone substrates, which are involved in the regulation of metabolism, differentiation, proliferation, senescence, protein degradation and apoptosis. 8 Overexpression of SIRT1 provides a cell su...

Cytokines control a variety of cellular responses including proliferation, differentiation, survival and functional activation, via binding to specific receptors expressed on the surface of target cells [1]. The cytokine receptors of the haemopoietin family are defined by the presence of a conserved 200 amino acid extracellular domain known as the haemopoietin domain [2]. We report here the isolation of NR6, a haemopoietin receptor that, like the p40 subunit of interleukin-12 (IL-12) [3] and the EBI3 gene induced by Epstein-Barr virus infection in lymphocytes [4], contains a typical haemopoietin domain but lacks transmembrane and cytoplasmic domains. Although in situ hybridisation revealed NR6 expression at multiple sites in the developing embryo, mice lacking NR6 did not display obvious abnormalities and were born in the expected numbers. Neonatal NR6(-/-) mice failed to suckle, however, and died within 24 hours of birth, suggesting that NR6 is necessary for the recognition or processing of pheromonal signals or for the mechanics of suckling itself. In addition, NR6(-/-) mice had reduced numbers of haemopoietic progenitor cells, suggesting a potential role in the regulation of primitive haemopoiesis.