Mutant HRAS as novel target for MEK and mTOR inhibitors

HRAS is a frequently mutated oncogene in cancer. However, mutant HRAS as drug target has not been investigated so far. Here, we show that mutant HRAS hyperactivates the RAS and the mTOR pathway in various cancer cell lines including lung, bladder and esophageal cancer. HRAS mutation sensitized toward growth inhibition by the MEK inhibitors AZD6244, MEK162 and PD0325901. Further, we found that MEK inhibitors induce apoptosis in mutant HRAS cell lines but not in cell lines lacking RAS mutations. In addition, knockdown of HRAS by siRNA blocked cell growth in mutant HRAS cell lines. Inhibition of the PI3K pathway alone or in combination with MEK inhibitors did not alter signaling nor had an impact on viability. However, inhibition of mTOR or combined inhibition of MEK and mTOR reduced cell growth in a synergistic manner. Finally, Ba/F3 cells transformed with mutant HRAS isoforms Q61L, Q61R and G12V demonstrated equal sensitivity towards MEK and mTOR inhibition. Our results show that HRAS mutations in cancer activate the RAS and mTOR pathways which might serve as a therapeutic option for patients with HRAS mutant tumors

pH-Sensing G Protein-Coupled Receptor OGR1 (GPR68) Expression and Activation Increases in Intestinal Inflammation and Fibrosis

Local extracellular acidification occurs at sites of inflammation. Proton-sensing ovarian cancer G-protein-coupled receptor 1 (OGR1, also known as GPR68) responds to decreases in extracellular pH. Our previous studies show a role for OGR1 in the pathogenesis of mucosal inflammation, suggesting a link between tissue pH and immune responses. Additionally, pH-dependent signalling is associated with the progression of intestinal fibrosis. In this study, we aimed to investigate OGR1 expression and OGR1-mediated signalling in patients with inflammatory bowel disease (IBD). Our results show that OGR1 expression significantly increased in patients with IBD compared to non-IBD patients, as demonstrated by qPCR and immunohistochemistry (IHC). Paired samples from non-inflamed and inflamed intestinal areas of IBD patients showed stronger OGR1 IHC staining in inflamed mucosal segments compared to non-inflamed mucosa. IHC of human surgical samples revealed OGR1 expression in macrophages, granulocytes, endothelial cells, and fibroblasts. OGR1-dependent inositol phosphate (IP) production was significantly increased in CD14+ monocytes from IBD patients compared to healthy subjects. Primary human and murine fibroblasts exhibited OGR1-dependent IP formation, RhoA activation, F-actin, and stress fibre formation upon an acidic pH shift. OGR1 expression and signalling increases with IBD disease activity, suggesting an active role of OGR1 in the pathogenesis of IBD. Keywords: OGR1 (GPR68) expression and function; fibroblasts; fibrosis; inflammatory bowel disease; pH-sensing GPCR

Prdx6 Deficiency Ameliorates DSS Colitis: Relevance of Compensatory Antioxidant Mechanisms

Background and Aims An imbalance between cellular antioxidant defence system[s] and reactive oxygen species [ROS]-driven oxidative stress has been implicated in the pathogenesis of inflammatory bowel disease. Peroxiredoxin [PRDX] 6 contributes to an appropriate redox balance by clearing ROS and reducing peroxidized membrane phospholipids. We here studied the role of PRDX6 in acute and chronic dextran sodium sulphate [DSS]-induced colitis. Methods To investigate the impact of PRDX6 on intestinal inflammation, we used wild type [WT], Prdx6 knock-out mice [Prdx6-/-] and transgenic mice [Prdx6tg/tg], overexpressing Prdx6. Acute and chronic colitis was induced by DSS in WT, Prdx6-/- and Prdx6tg/tg mice. Colitis was evaluated by endoscopy, colon length, histopathological assessment and myeloperoxidase [MPO] activity. Changes in mRNA and protein expression of pro-inflammatory cytokines and antioxidant enzymes were evaluated by real-time quantitative polymerase chain reaction [RT-qPCR] and western blot. Total glutathione [GSH] levels in colon samples were determined. Results Prdx6-/- mice exposed to acute and chronic DSS showed a significant decrease in the clinical parameters and in colonic expression of pro-inflammatory cytokines compared with WT mice. mRNA expression of antioxidant enzymes in colon samples was significantly increased in Prdx6-/- compared with WT mice exposed to acute and chronic DSS. In addition, total GSH levels were increased in Prdx6-/- mice treated with DSS in comparison with WT. Overexpression of Prdx6 did not significantly influence acute and chronic colitis. Conclusions Our data indicate that a lack of the antioxidant enzyme PRDX6 protects against the development of acute and chronic experimental colitis and is associated with increased expression and function of other antioxidant enzymes, suggesting effective compensatory mechanisms

Activation of pH-sensing receptor OGR1 (GPR68) induces ER stress via the IRE1α/JNK pathway in an intestinal epithelial Cell model

Proton-sensing ovarian cancer G-protein coupled receptor (OGR1) plays an important role in pH homeostasis. Acidosis occurs at sites of intestinal inflammation and can induce endoplasmic reticulum (ER) stress and the unfolded protein response (UPR), an evolutionary mechanism that enables cells to cope with stressful conditions. ER stress activates autophagy, and both play important roles in gut homeostasis and contribute to the pathogenesis of inflammatory bowel disease (IBD). Using a human intestinal epithelial cell model, we investigated whether our previously observed protective effects of OGR1 deficiency in experimental colitis are associated with a differential regulation of ER stress, the UPR and autophagy. Caco-2 cells stably overexpressing OGR1 were subjected to an acidic pH shift. pH-dependent OGR1-mediated signalling led to a significant upregulation in the ER stress markers, binding immunoglobulin protein (BiP) and phospho-inositol required 1α (IRE1α), which was reversed by a novel OGR1 inhibitor and a c-Jun N-terminal kinase (JNK) inhibitor. Proton-activated OGR1-mediated signalling failed to induce apoptosis, but triggered accumulation of total microtubule-associated protein 1 A/1B-light chain 3, suggesting blockage of late stage autophagy. Our results show novel functions for OGR1 in the regulation of ER stress through the IRE1α-JNK signalling pathway, as well as blockage of autophagosomal degradation. OGR1 inhibition might represent a novel therapeutic approach in IBD

Hypoxia ameliorates intestinal inflammation through NLRP3/mTOR downregulation and autophagy activation

Hypoxia regulates autophagy and nucleotide-binding oligomerization domain receptor, pyrin domain containing (NLRP)3, two innate immune mechanisms linked by mutual regulation and associated to IBD. Here we show that hypoxia ameliorates inflammation during the development of colitis by modulating autophagy and mammalian target of rapamycin (mTOR)/NLRP3 pathway. Hypoxia significantly reduces tumor necrosis factor α, interleukin (IL)-6 and NLRP3 expression, and increases the turnover of the autophagy protein p62 in colon biopsies of Crohn's disease patients, and in samples from dextran sulfate sodium-treated mice and Il-10 -/- mice. In vitro, NF-κB signaling and NLRP3 expression are reduced through hypoxia-induced autophagy. We also identify NLRP3 as a novel binding partner of mTOR. Dimethyloxalylglycine-mediated hydroxylase inhibition ameliorates colitis in mice, downregulates NLRP3 and promotes autophagy. We suggest that hypoxia counteracts inflammation through the downregulation of the binding of mTOR and NLRP3 and activation of autophagy.Hypoxia and HIF-1α activation are protective in mouse models of colitis, and the latter regulates autophagy. Here Cosin-Roger et al. show that hypoxia ameliorates intestinal inflammation in Crohn's patients and murine colitis models by inhibiting mTOR/NLRP3 pathway and promoting autophagy

Anti-inflammatory Function of High-Density Lipoproteins via Autophagy of IκB KinaseSummary

Background & Aims: Plasma levels of high-density lipoprotein (HDL) cholesterol are frequently found decreased in patients with inflammatory bowel disease (IBD). Therefore, and because HDL exerts anti-inflammatory activities, we investigated whether HDL and its major protein component apolipoprotein A-I (apoA-I) modulate mucosal inflammatory responses in vitro and in vivo. Methods: The human intestinal epithelial cell line T84 was used as the in vitro model for measuring the effects of HDL on the expression and secretion of tumor necrosis factor (TNF), interleukin-8 (IL-8), and intracellular adhesion molecule (ICAM). Nuclear factor-κB (NF-κB)-responsive promoter activity was studied by dual luciferase reporter assays. Mucosal damage from colitis induced by dextran sodium sulphate (DSS) and 2,4,6-trinitrobenzenesulfonic acid (TNBS) was scored by colonoscopy and histology in apoA-I transgenic (Tg) and apoA-I knockout (KO) and wild-type (WT) mice. Myeloperoxidase (MPO) activity and TNF and ICAM expression were determined in intestinal tissue samples. Autophagy was studied by Western blot analysis, immunofluorescence, and electron microscopy. Results: HDL and apoA-I down-regulated TNF-induced mRNA expression of TNF, IL-8, and ICAM, as well as TNF-induced NF-κB-responsive promoter activity. DSS/TNBS-treated apoA-I KO mice displayed increased mucosal damage upon both colonoscopy and histology, increased intestinal MPO activity and mRNA expression of TNF and ICAM as compared with WT and apoA-I Tg mice. In contrast, apoA-I Tg mice showed less severe symptoms monitored by colonoscopy and MPO activity in both the DSS and TNBS colitis models. In addition, HDL induced autophagy, leading to recruitment of phosphorylated IκB kinase to the autophagosome compartment, thereby preventing NF-κB activation and induction of cytokine expression. Conclusions: Taken together, the in vitro and in vivo findings suggest that HDL and apoA-I suppress intestinal inflammation via autophagy and are potential therapeutic targets for the treatment of IBD. Keywords: Apolipoprotein A-I, Autophagy, Inflammatory Bowel Disease, NF-κ