Stress granules are not present in Kras mutant cancers and do not control tumor growth.

Stress granules (SG) are membraneless ribonucleoprotein-based cytoplasmic organelles that assemble in response to stress. Their formation is often associated with an almost global suppression of translation, and the aberrant assembly or disassembly of these granules has pathological implications in neurodegeneration and cancer. In cancer, and particularly in the presence of oncogenic KRAS mutations, in vivo studies concluded that SG increase the resistance of cancer cells to stress. Hence, SG have recently been considered a promising target for therapy. Here, starting from our observations that genes coding for SG proteins are stimulated during development of pancreatic ductal adenocarcinoma, we analyze the formation of SG during tumorigenesis. We resort to in vitro, in vivo and in silico approaches, using mouse models, human samples and human data. Our analyses do not support that SG are formed during tumorigenesis of KRAS-driven cancers, at least that their presence is not universal, leading us to propose that caution is required before considering SG as therapeutic targets.

Cardiovirus leader proteins retarget RSK kinases toward alternative substrates to perturb nucleocytoplasmic traffic.

Proteins from some unrelated pathogens, including small RNA viruses of the family Picornaviridae, large DNA viruses such as Kaposi sarcoma-associated herpesvirus and even bacteria of the genus Yersinia can recruit cellular p90-ribosomal protein S6 kinases (RSKs) through a common linear motif and maintain the kinases in an active state. On the one hand, pathogens' proteins might hijack RSKs to promote their own phosphorylation (direct target model). On the other hand, some data suggested that pathogens' proteins might dock the hijacked RSKs toward a third interacting partner, thus redirecting the kinase toward a specific substrate. We explored the second hypothesis using the Cardiovirus leader protein (L) as a paradigm. The L protein is known to trigger nucleocytoplasmic trafficking perturbation, which correlates with hyperphosphorylation of phenylalanine-glycine (FG)-nucleoporins (FG-NUPs) such as NUP98. Using a biotin ligase fused to either RSK or L, we identified FG-NUPs as primary partners of the L-RSK complex in infected cells. An L protein mutated in the central RSK-interaction motif was readily targeted to the nuclear envelope whereas an L protein mutated in the C-terminal domain still interacted with RSK but failed to interact with the nuclear envelope. Thus, L uses distinct motifs to recruit RSK and to dock the L-RSK complex toward the FG-NUPs. Using an analog-sensitive RSK2 mutant kinase, we show that, in infected cells, L can trigger RSK to use NUP98 and NUP214 as direct substrates. Our data therefore illustrate a novel virulence mechanism where pathogens' proteins hijack and retarget cellular protein kinases toward specific substrates, to promote their replication or to escape immunity.

Two isoprenylated flavonoids from Dorstenia psilurus activate AMPK, stimulate glucose uptake, inhibit glucose production and lower glycemia.

Root extracts of a Cameroon medicinal plant, Dorstenia psilurus, were purified by screening for AMP-activated protein kinase (AMPK) activation in incubated mouse embryo fibroblasts (MEFs). Two isoprenylated flavones that activated AMPK were isolated. Compound 1 was identified as artelasticin by high-resolution electrospray ionization mass spectrometry and 2D-NMR while its structural isomer, compound 2, was isolated for the first time and differed only by the position of one double bond on one isoprenyl substituent. Treatment of MEFs with purified compound 1 or compound 2 led to rapid and robust AMPK activation at low micromolar concentrations and increased the intracellular AMP:ATP ratio. In oxygen consumption experiments on isolated rat liver mitochondria, compound 1 and compound 2 inhibited complex II of the electron transport chain and in freeze-thawed mitochondria succinate dehydrogenase was inhibited. In incubated rat skeletal muscles, both compounds activated AMPK and stimulated glucose uptake. Moreover, these effects were lost in muscles pre-incubated with AMPK inhibitor SBI-0206965, suggesting AMPK dependency. Incubation of mouse hepatocytes with compound 1 or compound 2 led to AMPK activation, but glucose production was decreased in hepatocytes from both wild-type and AMPKß1-/- mice, suggesting that this effect was not AMPK-dependent. However, when administered intraperitoneally to high-fat diet-induced insulin-resistant mice, compound 1 and compound 2 had blood glucose-lowering effects. In addition, compound 1 and compound 2 reduced the viability of several human cancer cells in culture. The flavonoids we have identified could be a starting point for the development of new drugs to treat type 2 diabetes.

Saturated fatty acids induce NLRP3 activation in human macrophages through K+ efflux resulting from phospholipid saturation and Na, K-ATPase disruption.

NLRP3 inflammasome plays a key role in Western diet-induced systemic inflammation and was recently shown to mediate long-lasting trained immunity in myeloid cells. Saturated fatty acids (SFAs) are sterile triggers able to induce the assembly of the NLRP3 inflammasome in macrophages, leading to IL-1ß secretion while unsaturated ones (UFAs) prevent SFAs-mediated NLRP3 activation. Unlike previous studies using LPS-primed bone marrow derived macrophages, we do not see any ROS or IRE-1α involvement in SFAs-mediated NLRP3 activation in human monocytes-derived macrophages. Rather we show that SFAs need to enter the cells and to be activated into acyl-CoA to lead to NLRP3 activation in human macrophages. However, their ß-oxidation is dispensable. Instead, they are channeled towards phospholipids but redirected towards lipid droplets containing triacylglycerol in the presence of UFAs. Lipidomic analyses and Laurdan fluorescence experiments demonstrate that SFAs induce a dramatic saturation of phosphatidylcholine (PC) correlated with a loss of membrane fluidity, both events inhibited by UFAs. The silencing of CCTα, the key enzyme in PC synthesis, prevents SFA-mediated NLRP3 activation, demonstrating the essential role of the de novo PC synthesis. This SFA-induced membrane remodeling promotes a disruption of the plasma membrane Na, K-ATPase, instigating a K+ efflux essential and sufficient for NLRP3 activation. This work opens novel therapeutic avenues to interfere with Western diet-associated diseases such as those targeting the glycerolipid pathway.

HBP1 phosphorylation by AKT regulates its transcriptional activity and glioblastoma cell proliferation.

The HMG-box protein 1 (HBP1) is a transcriptional regulator and a potential tumor suppressor that controls cell proliferation, differentiation and oncogene-mediated senescence. In a previous study, we showed that AKT activation through the PI3K/AKT/FOXO pathway represses HBP1 expression at the transcriptional level in human fibroblasts as well as in cancer cell lines. In the present study, we investigated whether AKT could also regulate HBP1 directly. First, AKT1 phosphorylated recombinant human HBP1 in vitro on three conserved sites, Ser380, Thr484 and Ser509. In living cells, we confirmed the phosphorylation of HBP1 on residues 380 and 509 using phospho-specific antibodies. HBP1 phosphorylation was induced by growth factors, such as EGF or IGF-1, which activated AKT. Conversely, it was blocked by treatment of cells with an AKT inhibitor (MK-2206) or by AKT knockdown. Next, we observed that HBP1 transcriptional activity was strongly modified by mutating its phosphorylation sites. The regulation of target genes such as DNMT1, P47phox, p16INK4A and cyclin D1 was also affected. HBP1 had previously been shown to limit glioma cell growth. Accordingly, HBP1 silencing by small-hairpin RNA increased human glioblastoma cell proliferation. Conversely, HBP1 overexpression decreased cell growth and foci formation. This effect was amplified by mutations that prevented phosphorylation by AKT, and blunted by mutations that mimicked phosphorylation. In conclusion, our results suggest that HBP1 phosphorylation by AKT blocks its functions as transcriptional regulator and tumor suppressor.