Soluble mediators such as thrombin and sphingosine-1-phosphate regulate morphological changes in endothelial cells that affect vascular permeability and new blood vessel formation. Although these ligands activate a similar set of heterotrimeric G proteins, thrombin causes cell contraction and rounding whereas sphingosine-1-phosphate induces cell spreading and migration. A functional requirement for Rho family GTPases in the cytoskeletal responses to both ligands has been established, yet the dynamics of their regulation and additional signaling mechanisms that lead to such opposite effects remain poorly understood. Using a pull-down assay to monitor the activity of Rho GTPases in human umbilical vein endothelial cells, we find significant temporal and quantitative differences in RhoA and Rac1 activation. High levels of active RhoA rapidly accumulate in cells in response to thrombin whereas Rac1 is inhibited. In contrast, sphingosine-1-phosphate addition leads to comparatively weak and delayed activation of RhoA and it activates Rac1. In addition, we show here that sphingosine-1-phosphate treatment activates a Src family kinase and triggers recruitment of the F-actin-binding protein cortactin to sites of actin polymerization at the rim of membrane ruffles. Both Src and Rac pathways are essential for lamellipodia targeting of cortactin. Further, Src plays a determinant role in sphingosine-1-phosphate-induced cell spreading and migration. Taken together these data demonstrate that the thrombin-induced contractile and immobile phenotype in endothelial cells reflects both robust RhoA activation and Rac inhibition, whereas Src- and Rac-dependent events couple sphingosine-1-phosphate receptors to the actin polymerizing machinery that drives the extension of lamellipodia and cell migration.

It is noteworthy that bacterial or viral infections, and the resulting chronic inflammation, have been shown to predispose individuals to certain types of cancer. Remarkably, these microbes upregulated some transcription factors involved in the regulation of the epithelial to mesenchymal transition, referred herein as EMT. EMT is a cellular process that consists in the conversion of epithelial cell phenotype to a mesenchymal phenotype. Under physiological conditions EMT is clearly important for embryogenesis, organ development, wound repair and tissue remodeling. However, EMT may also be activated under pathologic conditions, more particularly in carcinogenesis and metastatic progression. In this review, we make a parallel between microbes- and growth factors-induced transcription factors. A unifying EMT model then emerges that may help in understanding the development of microbial pathogenesis and in defining new potential future therapeutic strategy in treating diseases linked to infections.

Colitis-associated cancer (CAC) is a complication of inflammatory bowel disease (IBD). Binding of extracellular ATP to the purinergic receptor P2RX7 has emerged as a critical event in controlling intestinal inflammation, acting to limit elevation of proinflammatory mast cells and cytokines and promote survival of regulatory T cells (Treg) and enteric neurons. In this study, we investigated the effect of P2RX7 blockade in an established mouse model of CAC. Using genetic and pharmacologic tools, we found unexpectedly that while P2RX7 mediated inflammatory responses, it also acted at an early time to suppress CAC development. P2RX7 blockade enhanced proliferation of intestinal epithelial cells and protected them from apoptosis. The proliferative effects of P2RX7 blockade were associated with an increased production of TGFβ1 that was sufficient to stimulate the proliferation of intestinal epithelial cells. Finally, P2RX7 blockade also altered immune cell infiltration and promoted Treg accumulation within lesions of the digestive system. Taken together, our findings reveal an unexpected role for P2RX7 in preventing CAC, suggesting cautions in the use of P2RX7 inhibitors to treat IBD given the possibility of increasing risks CAC as a result. Cancer Res; 75(5); 835–45. ©2015 AACR.

Only a subpopulation of non-small cell lung cancer (NSCLC) patients responds to immunotherapies, highlighting the urgent need to develop therapeutic strategies to improve patient outcome. We develop a chemical positive modulator (HEI3090) of the purinergic P2RX7 receptor that potentiates αPD-1 treatment to effectively control the growth of lung tumors in transplantable and oncogene-induced mouse models and triggers long lasting antitumor immune responses. Mechanistically, the molecule stimulates dendritic P2RX7-expressing cells to generate IL-18 which leads to the production of IFN-γ by Natural Killer and CD4⁺ T cells within tumors. Combined with immune checkpoint inhibitor, the molecule induces a complete tumor regression in 80% of LLC tumor-bearing mice. Cured mice are also protected against tumor re-challenge due to a CD8-dependent protective response. Hence, combination treatment of small-molecule P2RX7 activator followed by immune checkpoint inhibitor represents a strategy that may be active against NSCLC.

Microbes regulate a large panel of intracellular signalling events that can promote inflammation and/or enhance tumour progression. Indeed, it has been shown that infection of human intestinal cells with the Afa/Dr diffusely adhering E. coli C1845 strain induces expression of pro‐angiogenic and pro‐inflammatory genes. Here, we demonstrate that exposure of cryptic‐like intestinal epithelial cells to C1845 bacteria induces HIF‐1α protein levels. This effect depends on the binding of F1845 adhesin to the membrane‐associated DAF receptor that initiates signalling cascades promoting translational mechanisms. Indeed, inhibition of MAPK and PI‐3K decreases HIF‐1α protein levels and blocks C1845‐induced phosphorylation of the ribosomal S6 protein. Using RNA interference we show that bacteria‐induced HIF‐1α regulates the expression of IL‐8, VEGF and Twist1, thereby pointing to a role for HIF‐1 in angiogenesis and inflammation. In addition, infection correlates with a loss of E‐cadherin and cytokeratin 18 and a rise in fibronectin, suggesting that bacteria may induce an epithelial to mesenchymal transition‐like phenotype. Since HIF‐1α silencing results in reversion of bacteria‐induced EMT markers, we speculate that HIF‐1α plays a key role linking bacterial infection to angiogenesis, inflammation and some aspects of cancer initiation.

Invading bacteria are recognized, captured and killed by a specialized form of autophagy, called xenophagy. Recently, defects in xenophagy in Crohn’s disease (CD) have been implicated in the pathogenesis of human chronic inflammatory diseases of uncertain etiology of the gastrointestinal tract. We show here that pathogenic adherent-invasive Escherichia coli (AIEC) isolated from CD patients are able to adhere and invade neutrophils, which represent the first line of defense against bacteria. Of particular interest, AIEC infection of neutrophil-like PLB-985 cells blocked autophagy at the autolysosomal step, which allowed intracellular survival of bacteria and exacerbated interleukin-8 (IL-8) production. Interestingly, this block in autophagy correlated with the induction of autophagic cell death. Likewise, stimulation of autophagy by nutrient starvation or rapamycin treatment reduced intracellular AIEC survival and IL-8 production. Finally, treatment with an inhibitor of autophagy decreased cell death of AIEC-infected neutrophil-like PLB-985 cells. In conclusion, excessive autophagy in AIEC infection triggered cell death of neutrophils.