Members of the Rho GTPase family play a central role in the orchestration of cytoskeletal rearrangements, which are of prime importance in endothelial cell physiology. To explore their role in this specialized cell type, we used the bacterial toxin cytotoxic necrotizing factor 1 (CNF1) as a Rho GTPase activator. Punctate filamentous actin structures appeared along the ventral plasma membrane of endothelial cells and were identified as the core of podosomes by the distinctive vinculin ring around the F-actin. Rho, Rac, and Cdc42 were all identified as targets of CNF1, but only a constitutively active mutant of Cdc42 could substitute for CNF1 in podosome induction. Accordingly, organization of F-actin in these structures was highly dependent on the main Cdc42 cytoskeletal effector N-Wiskott-Aldrich syndrome protein. Other components of the actin machinery such as Arp2/3 and for the first time WIP also colocalized at these sites. Like CNF1 treatment, sustained Cdc42 activity induced a time-dependent F-actin-vinculin reorganization, prevented cytokinesis, and downregulated Rho activity. Finally, podosomes were also detected on endothelial cells explanted from patients undergoing cardiac surgery. These data provide the first description of podosomes in endothelial cells. The identification of such specialized structures opens up a new field of investigation in terms of endothelium pathophysiology.

Gastric cancer’s bad incidence, prognosis, cellular and molecular heterogeneity amongst others make this disease a major health issue worldwide. Understanding this affliction is a priority for proper patients’ management and for the development of efficient therapeutical strategies. This review gives an overview of major scientific advances, made during the past 5-years, to improve the comprehension of gastric adenocarcinoma. A focus was made on the different actors of gastric carcinogenesis, including, Helicobacter pylori cancer stem cells, tumour microenvironment and microbiota. New and recent potential biomarkers were assessed as well as emerging therapeutical strategies involving cancer stem cells targeting as well as immunotherapy. Finally, recent experimental models to study this highly complex disease were discussed, highlighting the importance of gastric cancer understanding in the hard-fought struggle against cancer relapse, metastasis and bad prognosis.

Cytoskeletal rearrangements are central to endothelial cell physiology and are controlled by soluble factors, matrix proteins, cell-cell interactions, and mechanical forces. We previously reported that aortic endothelial cells can rearrange their cytoskeletons into complex actin-based structures called podosomes when a constitutively active mutant of Cdc42 is expressed. We now report that transforming growth factor beta (TGF-beta) promotes podosome formation in primary aortic endothelial cells. TGF-beta-induced podosomes assembled together into large ring- or crescent-shaped structures. Their formation was dependent on protein synthesis and required functional Src, phosphatidylinositide 3-kinase, Cdc42, RhoA, and Smad signaling. MT1-MMP and metalloprotease 9 (MMP9), both upregulated by TGF-beta, were detected at sites of podosome formation, and MT1-MMP was found to be involved in the local degradation of extracellular matrix proteins beneath the podosomes and required for the invasion of collagen gels by endothelial cells. We propose that TGF-beta plays an important role in endothelial cell physiology by inducing the formation of podosomal structures endowed with metalloprotease activity that may contribute to arterial remodeling.

The involvement of Src, Cdc42, RhoA and PKC in the regulation of podosome assembly has been identified in various cell models. In endothelial cells, the ectopic expression of constitutively active mutants of Src or Cdc42, but not RhoA, induced the formation of podosomes. Short-term exposure to phorbol-12-myristate-13-acetate (PMA) induced the appearance of podosomes and rosettes after initial disruption of stress fibres. Molecular analysis of PMA-induced podosomes and rosettes revealed that their composition was identical to that of podosomes described in other models. Pharmacological inhibition and siRNA knock-down experiments revealed that both PKCα and PKCδ isotypes were necessary for podosome assembly. However, only constitutively active PKCα could mimic PMA in podosome formation. Src, Cdc42 and RhoA were required downstream of PKCs in this process. Src could be positioned between PKC and Cdc42 in a linear cascade leading to podosome assembly. Using in vitro matrix degradation assays, we demonstrated that PMA-induced podosomes are endowed with proteolytic activities involving MT1-MMP-mediated activation of MMP2. Endothelial podosomes may be involved in subendothelial matrix degradation during endothelium remodelling in pathophysiological processes.

Purpose: Gastric carcinomas are heterogeneous, and the current therapy remains essentially based on surgery with conventional chemotherapy and radiotherapy. This study aimed to characterize biomarkers allowing the detection of cancer stem cells (CSC) in human gastric carcinoma of different histologic types. Experimental Design: The primary tumors from 37 patients with intestinal- or diffuse-type noncardia gastric carcinoma were studied, and patient-derived tumor xenograft (PDX) models in immunodeficient mice were developed. The expressions of 10 putative cell surface markers of CSCs, as well as aldehyde dehydrogenase (ALDH) activity, were studied, and the tumorigenic properties of cells were evaluated by in vitro tumorsphere assays and in vivo xenografts by limiting dilution assays. Results: We found that a subpopulation of gastric carcinoma cells expressing EPCAM, CD133, CD166, CD44, and a high ALDH activity presented the properties to generate new heterogeneous tumorspheres in vitro and tumors in vivo. CD44 and CD166 were coexpressed, representing 6.1% to 37.5% of the cells; ALDH activity was detected in 1.6% to 15.4% of the cells; and the ALDH+ cells represented a core within the CD44+/CD166+ subpopulation that contained the highest frequency of tumorigenic CSCs in vivo. The ALDH+ cells possessed drug efflux properties and were more resistant to standard chemotherapy than the ALDH− cells, a process that was partially reversed by verapamil treatment. Conclusions: CD44 and ALDH are the most specific biomarkers to detect and isolate tumorigenic and chemoresistant gastric CSCs in noncardia gastric carcinomas independently of the histologic classification of the tumor. Clin Cancer Res; 23(6); 1586–97. ©2016 AACR.