Fascin is an actin-binding and bundling protein that is highly upregulated in most epithelial cancers. Fascin promotes cell migration and adhesion dynamics in vitro and tumour cell metastasis in vivo. However, potential non-actin bundling roles for fascin remain unknown. Here, we show for the first time that fascin can directly interact with the microtubule cytoskeleton and that this does not depend upon fascin-actin bundling. Microtubule binding contributes to fascin-dependent control of focal adhesion dynamics and cell migration speed. We also show that fascin forms a complex with focal adhesion kinase (FAK, also known as PTK2) and Src, and that this signalling pathway lies downstream of fascin–microtubule association in the control of adhesion stability. These findings shed light on new non actin-dependent roles for fascin and might have implications for the design of therapies to target fascin in metastatic disease.

CCM2–CCM3 interactions protect CCM2 and CCM3 from proteasomal degradation, and both CCM2 and CCM3 are required for normal endothelial cell network formation.

Dynamic modulation of endothelial cell-to-cell and cell–to–extracellular matrix (ECM) adhesion is essential for blood vessel patterning and functioning. Yet the molecular mechanisms involved in this process have not been completely deciphered. We identify the adhesion G protein–coupled receptor (ADGR) Latrophilin 2 (LPHN2) as a novel determinant of endothelial cell (EC) adhesion and barrier function. In cultured ECs, endogenous LPHN2 localizes at ECM contacts, signals through cAMP/Rap1, and inhibits focal adhesion (FA) formation and nuclear localization of YAP/TAZ transcriptional regulators, while promoting tight junction (TJ) assembly. ECs also express an endogenous LPHN2 ligand, fibronectin leucine-rich transmembrane 2 (FLRT2), that prevents ECM-elicited EC behaviors in an LPHN2-dependent manner. Vascular ECs of lphn2a knock-out zebrafish embryos become abnormally stretched, display a hyperactive YAP/TAZ pathway, and lack proper intercellular TJs. Consistently, blood vessels are hyperpermeable, and intravascularly injected cancer cells extravasate more easily in lphn2a null animals. Thus, LPHN2 ligands, such as FLRT2, may be therapeutically exploited to interfere with cancer metastatic dissemination.

Although subcellular positioning of endosomes significantly impacts on their functions, the molecular mechanisms governing the different steady-state distribution of early endosomes (EEs) and late endosomes (LEs)/lysosomes (LYs) in peripheral and perinuclear eukaryotic cell areas, respectively, are still unsolved. We unveil that such differences arise because, while LE retrograde transport depends on the dynein microtubule (MT) motor only, the one of EEs requires the cooperative antagonism of dynein and kinesin-14 KIFC1, a MT minus end-directed motor involved in cancer progression. Mechanistically, the Ser-x-Ile-Pro (SxIP) motif-mediated interaction of the endoplasmic reticulum transmembrane protein stromal interaction molecule 1 (STIM1) with the MT plus endbinding protein 1 (EB1) promotes its association with the p150Glued subunit of the dynein activator complex dynactin and the distinct location of EEs and LEs/LYs. The peripheral distribution of EEs requires their p150Glued-mediated simultaneous engagement with dynein and SxIP motif-containing KIFC1, via HOOK1 and HOOK3 adaptors, respectively. In sum, we provide evidence that distinct minus end-directed MT motor systems drive the differential transport and subcellular distribution of EEs and LEs in mammalian cells.

The formation of a functional blood vessel network relies on the ability of endothelial cells (ECs) to dynamically rearrange their adhesive contacts in response to blood flow and guidance cues, such as vascular endothelial growth factor-A (VEGF-A) and class 3 semaphorins (SEMA3s). Neuropilin 1 (NRP1) is essential for blood vessel development, independently of its ligands VEGF-A and SEMA3, through poorly understood mechanisms. Grounding on unbiased proteomic analysis, we report here that NRP1 acts as an endocytic chaperone primarily for adhesion receptors on the surface of unstimulated ECs. NRP1 localizes at adherens junctions (AJs) where, interacting with VE-cadherin, promotes its basal internalization-dependent turnover and favors vascular permeability initiated by histamine in both cultured ECs and mice. We identify a splice variant of tryptophanyl-tRNA synthetase (mini-WARS) as an unconventionally secreted extracellular inhibitory ligand of NRP1 that, by stabilizing it at the AJs, slows down both VE-cadherin turnover and histamine-elicited endothelial leakage. Thus, our work shows a role for NRP1 as a major regulator of AJs plasticity and reveals how mini-WARS acts as a physiological NRP1 inhibitory ligand in the control of VE-cadherin endocytic turnover and vascular permeability.

There is growing evidence that ion channels are critically involved in cancer cell invasiveness and metastasis. However, the molecular mechanisms of ion signaling promoting cancer behavior are poorly understood and the complexity of the underlying remodeling during metastasis remains to be explored. Here, using a variety of in vitro and in vivo techniques, we show that metastatic prostate cancer cells acquire a specific Na + /Ca 2+ signature required for persistent invasion. We identify the Na + leak channel, NALCN, which is overexpressed in metastatic prostate cancer, as a major initiator and regulator of Ca 2+ oscillations required for invadopodia formation. Indeed, NALCN-mediated Na + influx into cancer cells maintains intracellular Ca 2+ oscillations via a specific chain of ion transport proteins including plasmalemmal and mitochondrial Na + /Ca 2+ exchangers, SERCA and store-operated channels. This signaling cascade promotes activity of the NACLN-colocalized proto-oncogene Src kinase, actin remodeling and secretion of proteolytic enzymes, thus increasing cancer cell invasive potential and metastatic lesions in vivo. Overall, our findings provide new insights into an ion signaling pathway specific for metastatic cells where NALCN acts as persistent invasion controller.

Dynamic modulation of endothelial cell (EC) adhesion to extracellular matrix (ECM) in response to mechanostimuli is essential for blood vessel patterning and functioning. Yet, the molecular mechanisms involved in this biological process are far to be completely deciphered. Here, we identify the adhesion G protein-coupled receptor (ADGR) Latrophilin 2 (LPHN2) as a novel determinant of vascular morphogenesis and endothelial barrier function. In cultured ECs, endogenous LPHN2 localizes at ECM adhesions, signals through cAMP/Rap1, and, via its fibronectin-leucine-rich transmembrane (FLRT)-binding domain, negatively regulates ECM-elicited haptotaxis. ECs also express endogenous FLRT2 ligand that promotes cAMP/Rap1 signaling and hinders haptotaxis in a LPHN2-dependent manner. Vascular ECs of lphn2a knock-out zebrafish embryos become abnormally stretched, display a hyperactive Hippo mechanosensing pathway, and lack proper intercellular junctions. Indeed, intravascularly injected cancer cells extravasate more easily in lphn2a null animals. Thus, LPHN2 ligands, such as FLRT2, may be therapeutically exploited to interfere with cancer metastatic dissemination.

Many nervous proteins are expressed in cancer cells. In this report, we asked whether the synaptic protein neuroligin 1 (NLGN1) was expressed by prostatic and pancreatic carcinomas; in addition, given the tendency of these tumors to interact with nerves, we asked whether NLGN1 played a role in this process. Through immunohistochemistry on human tissue microarrays, we showed that NLGN1 is expressed by prostatic and pancreatic cancer tissues in discrete stages and tumor districts. Next, we performed in vitro and in vivo assays, demonstrating that NLGN1 promotes cancer cell invasion and migration along nerves. Because of the established role of the neurotrophic factor glial cell line-derived neurotrophic factor (GDNF) in tumor–nerve interactions, we assessed a potential NLGN1–GDNF cooperation. We found that blocking GDNF activity with a specific antibody completely inhibited NLGN1-induced in vitro cancer cell invasion of nerves. Finally, we demonstrated that, in the presence of NLGN1, GDNF markedly activates cofilin, a cytoskeletal regulatory protein, altering filopodia dynamics. In conclusion, our data further prove the existence of a molecular and functional cross-talk between the nervous system and cancer cells. NLGN1 was shown here to function along one of the most represented neurotrophic factors in the nerve microenvironment, possibly opening new therapeutic avenues.