Cell division-dependent dissemination following E-cadherin loss underlies initiation of diffuse-type gastric cancer.

Loss of the cell-cell adhesion protein E-cadherin underlies the development of diffuse-type gastric cancer (DGC), which is characterized by the gradual accumulation of tumor cells originating from the gastric epithelium in the surrounding stroma. How E-cadherin deficiency drives DGC formation remains elusive. Therefore, we investigated the consequences of E-cadherin loss on gastric epithelial organization utilizing a human gastric organoid model and histological analyses of early-stage DGC lesions. E-cadherin depletion from gastric organoids recapitulates DGC initiation, with progressive loss of a single-layered architecture and detachment of individual cells. We found that E-cadherin deficiency in gastric epithelia does not lead to a general loss of epithelial cohesion but disrupts the spindle orientation machinery. This leads to a loss of planar cell division orientation and, consequently, daughter cells are positioned outside of the gastric epithelial layer. Although basally delaminated cells fail to detach and instead reintegrate into the epithelium, apically mispositioned daughter cells can trigger the gradual loss of the single-layered epithelial architecture. This impaired architecture hampers reintegration of mispositioned daughter cells and enables basally delaminated cells to disseminate into the surrounding matrix. Taken together, our findings describe how E-cadherin deficiency disrupts gastric epithelial architecture through displacement of dividing cells and provide new insights in the onset of DGC. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

A mechanical G2 checkpoint controls epithelial cell division through E-cadherin-mediated regulation of Wee1-Cdk1.

Epithelial cell divisions are coordinated with cell loss to preserve epithelial integrity. However, how epithelia adapt their rate of cell division to changes in cell number, for instance during homeostatic turnover or wounding, is not well understood. Here, we show that epithelial cells sense local cell density through mechanosensitive E-cadherin adhesions to control G2/M cell-cycle progression. As local cell density increases, tensile forces on E-cadherin adhesions are reduced, which prompts the accumulation of the G2 checkpoint kinase Wee1 and downstream inhibitory phosphorylation of Cdk1. Consequently, dense epithelia contain a pool of cells that are temporarily halted in G2 phase. These cells are readily triggered to divide following epithelial wounding due to the consequent increase in intercellular forces and resulting degradation of Wee1. Our data collectively show that epithelial cell division is controlled by a mechanical G2 checkpoint, which is regulated by cell-density-dependent intercellular forces sensed and transduced by E-cadherin adhesions.

An asymmetric junctional mechanoresponse coordinates mitotic rounding with epithelial integrity.

Epithelia are continuously self-renewed, but how epithelial integrity is maintained during the morphological changes that cells undergo in mitosis is not well understood. Here, we show that as epithelial cells round up when they enter mitosis, they exert tensile forces on neighboring cells. We find that mitotic cell-cell junctions withstand these tensile forces through the mechanosensitive recruitment of the actin-binding protein vinculin to cadherin-based adhesions. Surprisingly, vinculin that is recruited to mitotic junctions originates selectively from the neighbors of mitotic cells, resulting in an asymmetric composition of cadherin junctions. Inhibition of junctional vinculin recruitment in neighbors of mitotic cells results in junctional breakage and weakened epithelial barrier. Conversely, the absence of vinculin from the cadherin complex in mitotic cells is necessary to successfully undergo mitotic rounding. Our data thus identify an asymmetric mechanoresponse at cadherin adhesions during mitosis, which is essential to maintain epithelial integrity while at the same time enable the shape changes of mitotic cells.

Multiple Rap1 effectors control Epac1-mediated tightening of endothelial junctions.

Epac1 and Rap1 mediate cAMP-induced tightening of endothelial junctions. We have previously found that one of the mechanisms is the inhibition of Rho-mediated tension in radial stress fibers by recruiting the RhoGAP ArhGAP29 in a complex containing the Rap1 effectors Rasip1 and Radil. However, other mechanisms have been proposed as well, most notably the induction of tension in circumferential actin cables by Cdc42 and its GEF FGD5. Here, we have investigated how Rap1 controls FGD5/Cdc42 and how this interconnects with Radil/Rasip1/ArhGAP29. Using endothelial barrier measurements, we show that Rho inhibition is not sufficient to explain the barrier stimulating effect of Rap1. Indeed, Cdc42-mediated tension is induced at cell-cell contacts upon Rap1 activation and this is required for endothelial barrier function. Depletion of potential Rap1 effectors identifies AF6 to mediate Rap1 enhanced tension and concomitant Rho-independent barrier function. When overexpressed in HEK293T cells, AF6 is found in a complex with FGD5 and Radil. From these results we conclude that Rap1 utilizes multiple pathways to control tightening of endothelial junctions, possibly through a multiprotein effector complex, in which AF6 functions to induce tension in circumferential actin cables.

The small GTPase RALA controls c-Jun N-terminal kinase-mediated FOXO activation by regulation of a JIP1 scaffold complex.

FOXO (forkhead box O) transcription factors are tumor suppressors and increase the life spans of model organisms. Cellular stress, in particular oxidative stress caused by an increase in levels of reactive oxygen species (ROS), activates FOXOs through JNK-mediated phosphorylation. Importantly, JNK regulation of FOXO is evolutionarily conserved. Here we identified the pathway that mediates ROS-induced JNK-dependent FOXO regulation. Following increased ROS, RALA is activated by the exchange factor RLF (RalGDS-like factor), which is in complex with JIP1 (C-Jun-amino-terminal-interacting protein 1) and JNK. Active RALA consequently regulates assembly and activation of MLK3, MKK4, and JNK onto the JIP1 scaffold. Furthermore, regulation of FOXO by RALA and JIP1 is conserved in C. elegans, where both ral-1 and jip-1 depletion impairs heat shock-induced nuclear translocation of the FOXO orthologue DAF16.

Rap1 can bypass the FAK-Src-Paxillin cascade to induce cell spreading and focal adhesion formation.

We developed new image analysis tools to analyse quantitatively the extracellular-matrix-dependent cell spreading process imaged by live-cell epifluorescence microscopy. Using these tools, we investigated cell spreading induced by activation of the small GTPase, Rap1. After replating and initial adhesion, unstimulated cells exhibited extensive protrusion and retraction as their spread area increased, and displayed an angular shape that was remodelled over time. In contrast, activation of endogenous Rap1, via 007-mediated stimulation of Epac1, induced protrusion along the entire cell periphery, resulting in a rounder spread surface, an accelerated spreading rate and an increased spread area compared to control cells. Whereas basal, anisotropic, spreading was completely dependent on Src activity, Rap1-induced spreading was refractory to Src inhibition. Under Src inhibited conditions, the characteristic Src-induced tyrosine phosphorylations of FAK and paxillin did not occur, but Rap1 could induce the formation of actomyosin-connected adhesions, which contained vinculin at levels comparable to that found in unperturbed focal adhesions. From these results, we conclude that Rap1 can induce cell adhesion and stimulate an accelerated rate of cell spreading through mechanisms that bypass the canonical FAK-Src-Paxillin signalling cascade.

Rap2A links intestinal cell polarity to brush border formation.

The microvillus brush border at the apex of the highly polarized enterocyte allows the regulated uptake of nutrients from the intestinal lumen. Here, we identify the small G protein Rap2A as a molecular link that couples the formation of microvilli directly to the preceding cell polarization. Establishment of apicobasal polarity, which can be triggered by the kinase LKB1 in single, isolated colon cells, results in enrichment of PtdIns(4,5)P(2) at the apical membrane. The subsequent recruitment of phospholipase D1 allows polarized accumulation of phosphatidic acid, which provides a local cue for successive signalling by the guanine nucleotide exchange factor PDZGEF, the small G protein Rap2A, its effector TNIK, the kinase MST4 and, ultimately, the actin-binding protein Ezrin. Thus, epithelial cell polarization is translated directly into the acquisition of brush borders through a small G protein signalling module whose action is positioned by a cortical lipid cue.

Role of Epac and protein kinase A in thyrotropin-induced gene expression in primary thyrocytes.

cAMP pathway activation by thyrotropin (TSH) induces differentiation and gene expression in thyrocytes. We investigated which partners of the cAMP cascade regulate gene expression modulations: protein kinase A and/or the exchange proteins directly activated by cAMP (Epac). Human primary cultured thyrocytes were analysed by microarrays after treatment with the adenylate cyclase activator forskolin, the protein kinase A (PKA) activator 6-MB-cAMP and the Epac-selective cAMP analog 8-pCPT-2'-O-Me-cAMP (007) alone or combined with 6-MB-cAMP. Profiles were compared to those of TSH. Cultures treated with the adenylate cyclase- or the PKA activator alone or the latter combined with 007 had profiles similar to those induced by TSH. mRNA profiles of 007-treated cultures were highly distinct from TSH-treated cells, suggesting that TSH-modulated gene expressions are mainly modulated by cAMP and PKA and not through Epac in cultured human thyroid cells. To investigate whether the Epac-Rap-RapGAP pathway could play a potential role in thyroid tumorigenesis, the mRNA expressions of its constituent proteins were investigated in two malignant thyroid tumor types. Modulations of this pathway suggest an increased Rap pathway activity in these cancers independent from cAMP activation.

The nucleoporin RanBP2 tethers the cAMP effector Epac1 and inhibits its catalytic activity.

Cyclic adenosine monophosphate (cAMP) is a second messenger that relays a wide range of hormone responses. In this paper, we demonstrate that the nuclear pore component RanBP2 acts as a negative regulator of cAMP signaling through Epac1, a cAMP-regulated guanine nucleotide exchange factor for Rap. We show that Epac1 directly interacts with the zinc fingers (ZNFs) of RanBP2, tethering Epac1 to the nuclear pore complex (NPC). RanBP2 inhibits the catalytic activity of Epac1 in vitro by binding to its catalytic CDC25 homology domain. Accordingly, cellular depletion of RanBP2 releases Epac1 from the NPC and enhances cAMP-induced Rap activation and cell adhesion. Epac1 also is released upon phosphorylation of the ZNFs of RanBP2, demonstrating that the interaction can be regulated by posttranslational modification. These results reveal a novel mechanism of Epac1 regulation and elucidate an unexpected link between the NPC and cAMP signaling.

Spatial regulation of cyclic AMP-Epac1 signaling in cell adhesion by ERM proteins.

Epac1 is a guanine nucleotide exchange factor for the small G protein Rap and is involved in membrane-localized processes such as integrin-mediated cell adhesion and cell-cell junction formation. Cyclic AMP (cAMP) directly activates Epac1 by release of autoinhibition and in addition induces its translocation to the plasma membrane. Here, we show an additional mechanism of Epac1 recruitment, mediated by activated ezrin-radixin-moesin (ERM) proteins. Epac1 directly binds with its N-terminal 49 amino acids to ERM proteins in their open conformation. Receptor-induced activation of ERM proteins results in increased binding of Epac1 and consequently the clustered localization of Epac1 at the plasma membrane. Deletion of the N terminus of Epac1, as well as disruption of the Epac1-ERM interaction by an interfering radixin mutant or small interfering RNA (siRNA)-mediated depletion of the ERM proteins, impairs Epac1-mediated cell adhesion. We conclude that ERM proteins are involved in the spatial regulation of Epac1 and cooperate with cAMP- and Rap-mediated signaling to regulate adhesion to the extracellular matrix.

The RapGEF PDZ-GEF2 is required for maturation of cell-cell junctions.

The small G-protein Rap1 is a critical regulator of cell-cell contacts and is activated by the remodeling of adherens junctions. Here we identify the Rap1 guanine nucleotide exchange factor PDZ-GEF2 as an upstream activator of Rap1 required for the maturation of adherens junctions in the lung carcinoma cells A549. Knockdown of PDZ-GEF2 results in the persistence of adhesion zippers at cell-cell contacts. Activation of Rap1A rescues junction maturation in absence of PDZ-GEF2, demonstrating that Rap1A is downstream of PDZ-GEF2 in this process. Moreover, depletion of Rap1A, but not Rap1B, impairs adherens junction maturation. siRNA for PDZ-GEF2 also lowers the levels of E-cadherin, an effect that can be mimicked by Rap1B, but not Rap1A siRNA. Since junctions in Rap1B depleted cells have a mature appearance, these data suggest that PDZ-GEF2 activates Rap1A and Rap1B to perform different functions. Our results present the first direct evidence that PDZ-GEF2 plays a critical role in the maturation of adherens junctions.