A YAP-centered mechanotransduction loop drives collective breast cancer cell invasion.

Dense and aligned Collagen I fibers are associated with collective cancer invasion led by protrusive tumor cells, leader cells. In some breast tumors, a population of cancer cells (basal-like cells) maintain several epithelial characteristics and express the myoepithelial/basal cell marker Keratin 14 (K14). Emergence of leader cells and K14 expression are regarded as interconnected events triggered by Collagen I, however the underlying mechanisms remain unknown. Using breast carcinoma organoids, we show that Collagen I drives a force-dependent loop, specifically in basal-like cancer cells. The feed-forward loop is centered around the mechanotransducer Yap and independent of K14 expression. Yap promotes a transcriptional program that enhances Collagen I alignment and tension, which further activates Yap. Active Yap is detected in invading breast cancer cells in patients and required for collective invasion in 3D Collagen I and in the mammary fat pad of mice. Our work uncovers an essential function for Yap in leader cell selection during collective cancer invasion.

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.

Co-cultures of colon cancer cells and cancer-associated fibroblasts recapitulate the aggressive features of mesenchymal-like colon cancer.

Background: Poor prognosis in colon cancer is associated with a high content of cancer-associated fibroblasts (CAFs) and an immunosuppressive tumor microenvironment. The relationship between these two features is incompletely understood. Here, we aimed to generate a model system for studying the interaction between cancer cells and CAFs and their effect on immune-related cytokines and T cell proliferation. Methods: CAFs were isolated from colon cancer liver metastases and were immortalized to prolong lifespan and improve robustness and reproducibility. Established medium and matrix compositions that support the growth of patient-derived organoids were adapted to also support CAF growth. Changes in growth pattern and cellular re-organization were assessed by confocal microscopy, live cell imaging, and immunofluorescence. Single cell RNA sequencing was used to study CAF/organoid co-culture-induced phenotypic changes in both cell types. Conditioned media were used to quantify the production of immunosuppressive factors and to assess their effect on T cell proliferation. Results: We developed a co-culture system in which colon cancer organoids and CAFs spontaneously organize into superstructures with a high capacity to contract and stiffen the extracellular matrix (ECM). CAF-produced collagen IV provided a basement membrane supporting cancer cell organization into glandular structures, reminiscent of human cancer histology. Single cell RNA sequencing analysis showed that CAFs induced a partial epithelial-to-mesenchymal-transition in a subpopulation of cancer cells, similar to what is observed in the mesenchymal-like consensus molecular subtype 4 (CMS4) colon cancer. CAFs in co-culture were characterized by high expression of ECM components, ECM-remodeling enzymes, glycolysis, hypoxia, and genes involved in immunosuppression. An expression signature derived from CAFs in co-culture identified a subpopulation of glycolytic myofibroblasts specifically residing in CMS1 and CMS4 colon cancer. Medium conditioned by co-cultures contained high levels of the immunosuppressive factors TGFß1, VEGFA and lactate, and potently inhibited T cell proliferation. Conclusion: Co-cultures of organoids and immortalized CAFs recapitulate the histological, biophysical, and immunosuppressive features of aggressive mesenchymal-like human CRC. The model can be used to study the mechanisms of immunosuppression and to test therapeutic strategies targeting the cross-talk between CAFs and cancer cells. It can be further modified to represent distinct colon cancer subtypes and (organ-specific) microenvironments.

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.

Liver Colonization by Colorectal Cancer Metastases Requires YAP-Controlled Plasticity at the Micrometastatic Stage.

Micrometastases of colorectal cancer can remain dormant for years prior to the formation of actively growing, clinically detectable lesions (i.e., colonization). A better understanding of this step in the metastatic cascade could help improve metastasis prevention and treatment. Here we analyzed liver specimens of patients with colorectal cancer and monitored real-time metastasis formation in mouse livers using intravital microscopy to reveal that micrometastatic lesions are devoid of cancer stem cells (CSC). However, lesions that grow into overt metastases demonstrated appearance of de novo CSCs through cellular plasticity at a multicellular stage. Clonal outgrowth of patient-derived colorectal cancer organoids phenocopied the cellular and transcriptomic changes observed during in vivo metastasis formation. First, formation of mature CSCs occurred at a multicellular stage and promoted growth. Conversely, failure of immature CSCs to generate more differentiated cells arrested growth, implying that cellular heterogeneity is required for continuous growth. Second, early-stage YAP activity was required for the survival of organoid-forming cells. However, subsequent attenuation of early-stage YAP activity was essential to allow for the formation of cell type heterogeneity, while persistent YAP signaling locked micro-organoids in a cellularly homogenous and growth-stalled state. Analysis of metastasis formation in mouse livers using single-cell RNA sequencing confirmed the transient presence of early-stage YAP activity, followed by emergence of CSC and non-CSC phenotypes, irrespective of the initial phenotype of the metastatic cell of origin. Thus, establishment of cellular heterogeneity after an initial YAP-controlled outgrowth phase marks the transition to continuously growing macrometastases. SIGNIFICANCE: Characterization of the cell type dynamics, composition, and transcriptome of early colorectal cancer liver metastases reveals that failure to establish cellular heterogeneity through YAP-controlled epithelial self-organization prohibits the outgrowth of micrometastases. See related commentary by LeBleu, p. 1870.

Diffuse gastric cancer: Emerging mechanisms of tumor initiation and progression.

Gastric cancer is globally the fourth leading cause of cancer-related deaths. Patients with diffuse-type gastric cancer (DGC) particularly have a poor prognosis that only marginally improved over the last decades, as conventional chemotherapies are frequently ineffective and specific therapies are unavailable. Early-stage DGC is characterized by intramucosal lesions of discohesive cells, which can be present for many years before the emergence of advanced DGC consisting of highly proliferative and invasive cells. The mechanisms underlying the key steps of DGC development and transition to aggressive tumors are starting to emerge. Novel mouse and organoid models for DGC, together with multi-omic analyses of DGC tumors, revealed contributions of both tumor cell-intrinsic alterations and gradual changes in the tumor microenvironment to DGC progression. In this review, we will discuss how these recent findings are leading towards an understanding of the cellular and molecular mechanisms responsible for DGC initiation and malignancy, which may provide opportunities for targeted therapies.

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.

DLC1 is a direct target of activated YAP/TAZ that drives collective migration and sprouting angiogenesis.

Endothelial YAP/TAZ (YAP is also known as YAP1, and TAZ as WWTR1) signaling is crucial for sprouting angiogenesis and vascular homeostasis. However, the underlying molecular mechanisms that explain how YAP/TAZ control the vasculature remain unclear. This study reveals that the focal adhesion protein deleted-in-liver-cancer 1 (DLC1) is a direct transcriptional target of the activated YAP/TAZ-TEAD complex. We find that substrate stiffening and VEGF stimuli promote expression of DLC1 in endothelial cells. In turn, DLC1 expression levels are YAP and TAZ dependent, and constitutive activation of YAP is sufficient to drive DLC1 expression. DLC1 is needed to limit F-actin fiber formation, integrin-based focal adhesion lifetime and integrin-mediated traction forces. Depletion of endothelial DLC1 strongly perturbs cell polarization in directed collective migration and inhibits the formation of angiogenic sprouts. Importantly, ectopic expression of DLC1 is sufficient to restore migration and angiogenic sprouting in YAP-depleted cells. Together, these findings point towards a crucial and prominent role for DLC1 in YAP/TAZ-driven endothelial adhesion remodeling and collective migration during angiogenesis.This article has an associated First Person interview with the first author of the paper.

Adhesion to the host cell surface is sufficient to mediate Listeria monocytogenes entry into epithelial cells.

The intestinal epithelium is the first physiological barrier breached by the Gram-positive facultative pathogen Listeria monocytogenes during an in vivo infection. Listeria monocytogenes binds to the epithelial host cell receptor E-cadherin, which mediates a physical link between the bacterium and filamentous actin (F-actin). However, the importance of anchoring the bacterium to F-actin through E-cadherin for bacterial invasion has not been tested directly in epithelial cells. Here we demonstrate that depleting αE-catenin, which indirectly links E-cadherin to F-actin, did not decrease L. monocytogenes invasion of epithelial cells in tissue culture. Instead, invasion increased due to increased bacterial adhesion to epithelial monolayers with compromised cell-cell junctions. Furthermore, expression of a mutant E-cadherin lacking the intracellular domain was sufficient for efficient L. monocytogenes invasion of epithelial cells. Importantly, direct biotin-mediated binding of bacteria to surface lipids in the plasma membrane of host epithelial cells was sufficient for uptake. Our results indicate that the only requirement for L. monocytogenes invasion of epithelial cells is adhesion to the host cell surface, and that E-cadherin-mediated coupling of the bacterium to F-actin is not required.

E-cadherin and LGN align epithelial cell divisions with tissue tension independently of cell shape.

Tissue morphogenesis requires the coordinated regulation of cellular behavior, which includes the orientation of cell division that defines the position of daughter cells in the tissue. Cell division orientation is instructed by biochemical and mechanical signals from the local tissue environment, but how those signals control mitotic spindle orientation is not fully understood. Here, we tested how mechanical tension across an epithelial monolayer is sensed to orient cell divisions. Tension across Madin-Darby canine kidney cell monolayers was increased by a low level of uniaxial stretch, which oriented cell divisions with the stretch axis irrespective of the orientation of the cell long axis. We demonstrate that stretch-induced division orientation required mechanotransduction through E-cadherin cell-cell adhesions. Increased tension on the E-cadherin complex promoted the junctional recruitment of the protein LGN, a core component of the spindle orientation machinery that binds the cytosolic tail of E-cadherin. Consequently, uniaxial stretch triggered a polarized cortical distribution of LGN. Selective disruption of trans engagement of E-cadherin in an otherwise cohesive cell monolayer, or loss of LGN expression, resulted in randomly oriented cell divisions in the presence of uniaxial stretch. Our findings indicate that E-cadherin plays a key role in sensing polarized tensile forces across the tissue and transducing this information to the spindle orientation machinery to align cell divisions.

Cell division orientation is coupled to cell-cell adhesion by the E-cadherin/LGN complex.

Both cell-cell adhesion and oriented cell division play prominent roles in establishing tissue architecture, but it is unclear how they might be coordinated. Here, we demonstrate that the cell-cell adhesion protein E-cadherin functions as an instructive cue for cell division orientation. This is mediated by the evolutionarily conserved LGN/NuMA complex, which regulates cortical attachments of astral spindle microtubules. We show that LGN, which adopts a three-dimensional structure similar to cadherin-bound catenins, binds directly to the E-cadherin cytosolic tail and thereby localizes at cell-cell adhesions. On mitotic entry, NuMA is released from the nucleus and competes LGN from E-cadherin to locally form the LGN/NuMA complex. This mediates the stabilization of cortical associations of astral microtubules at cell-cell adhesions to orient the mitotic spindle. Our results show how E-cadherin instructs the assembly of the LGN/NuMA complex at cell-cell contacts, and define a mechanism that couples cell division orientation to intercellular adhesion.

cAMP regulates DEP domain-mediated binding of the guanine nucleotide exchange factor Epac1 to phosphatidic acid at the plasma membrane.

Epac1 is a cAMP-regulated guanine nucleotide exchange factor for the small G protein Rap. Upon cAMP binding, Epac1 undergoes a conformational change that results in its release from autoinhibition. In addition, cAMP induces the translocation of Epac1 from the cytosol to the plasma membrane. This relocalization of Epac1 is required for efficient activation of plasma membrane-located Rap and for cAMP-induced cell adhesion. This translocation requires the Dishevelled, Egl-10, Pleckstrin (DEP) domain, but the molecular entity that serves as the plasma membrane anchor and the possible mechanism of regulated binding remains elusive. Here we show that Epac1 binds directly to phosphatidic acid. Similar to the cAMP-induced Epac1 translocation, this binding is regulated by cAMP and requires the DEP domain. Furthermore, depletion of phosphatidic acid by inhibition of phospholipase D1 prevents cAMP-induced translocation of Epac1 as well as the subsequent activation of Rap at the plasma membrane. Finally, mutation of a single basic residue within a polybasic stretch of the DEP domain, which abolishes translocation, also prevents binding to phosphatidic acid. From these results we conclude that cAMP induces a conformational change in Epac1 that enables DEP domain-mediated binding to phosphatidic acid, resulting in the tethering of Epac1 at the plasma membrane and subsequent activation of Rap.

Regulating Rap small G-proteins in time and space.

Signaling by the small G-protein Rap is under tight regulation by its GEFs and GAPs. These are multi-domain proteins that are themselves controlled by distinct upstream pathways, and thus couple different extra- and intracellular cues to Rap. The individual RapGEFs and RapGAPs are, in addition, targeted to specific cellular locations by numerous anchoring mechanisms and, consequently, may control different pools of Rap. Here, we review the various activating signals and targeting mechanisms of these proteins and discuss their contribution to the spatiotemporal regulation and biological functions of the Rap proteins.

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.

Ezrin is required for efficient Rap1-induced cell spreading.

The Rap family of small GTPases regulate the adhesion of cells to extracellular matrices. Several Rap-binding proteins have been shown to function as effectors that mediate Rap-induced adhesion. However, little is known regarding the relationships between these effectors, or about other proteins that are downstream of or act in parallel to the effectors. To establish whether an array of effectors was required for Rap-induced cell adhesion and spreading, and to find new components involved in Rap-signal transduction, we performed a small-scale siRNA screen in A549 lung epithelial cells. Of the Rap effectors tested, only Radil blocked Rap-induced spreading. Additionally, we identified a novel role for Ezrin downstream of Rap1. Ezrin was necessary for Rap-induced cell spreading, but not Rap-induced cell adhesion or basal adhesion processes. Furthermore, Ezrin depletion inhibited Rap-induced cell spreading in several cell lines, including primary human umbilical vein endothelial cells. Interestingly, Radixin and Moesin, two proteins with high homology to Ezrin, are not required for Rap-induced cell spreading and cannot compensate for loss of Ezrin to rescue Rap-induced cell spreading. Here, we present a novel function for Ezrin in Rap1-induced cell spreading and evidence of a non-redundant role of an ERM family member.

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.

Epac: defining a new mechanism for cAMP action.

cAMP is a second messenger that is essential for relaying hormonal responses in many biological processes. The discovery of the cAMP target Epac explained various effects of cAMP that could not be attributed to the established targets PKA and cyclic nucleotide-gated ion channels. Epac1 and Epac2 function as guanine nucleotide exchange factors for the small G protein Rap. cAMP analogs that selectively activate Epac have helped to reveal a role for Epac in processes ranging from insulin secretion to cardiac contraction and vascular permeability. Advances in the understanding of the activation mechanism of Epac and its regulation by diverse anchoring mechanisms have helped to elucidate the means by which cAMP fulfills these functions via Epac.

Direct spatial control of Epac1 by cyclic AMP.

Epac1 is a guanine nucleotide exchange factor (GEF) for the small G protein Rap and is directly activated by cyclic AMP (cAMP). Upon cAMP binding, Epac1 undergoes a conformational change that allows the interaction of its GEF domain with Rap, resulting in Rap activation and subsequent downstream effects, including integrin-mediated cell adhesion and cell-cell junction formation. Here, we report that cAMP also induces the translocation of Epac1 toward the plasma membrane. Combining high-resolution confocal fluorescence microscopy with total internal reflection fluorescence and fluorescent resonance energy transfer assays, we observed that Epac1 translocation is a rapid and reversible process. This dynamic redistribution of Epac1 requires both the cAMP-induced conformational change as well as the DEP domain. In line with its translocation, Epac1 activation induces Rap activation predominantly at the plasma membrane. We further show that the translocation of Epac1 enhances its ability to induce Rap-mediated cell adhesion. Thus, the regulation of Epac1-Rap signaling by cAMP includes both the release of Epac1 from autoinhibition and its recruitment to the plasma membrane.

P18 is a tumor suppressor gene involved in human medullary thyroid carcinoma and pheochromocytoma development.

In multiple endocrine neoplasia syndrome Type 2 (MEN2), medullary thyroid carcinoma (MTC) and pheochromocytoma (PC) are associated with hereditary activating germ-line mutations in the RET proto-oncogene. Also in a large percentage of sporadic MTCs and PCs, somatic RET mutations appear to be involved in tumor formation. In one single MEN2 family an extensive variety in disease expression may be observed, indicating that additional genetic events are responsible for progression of the disease towards a more aggressive phenotype. However, these additional mutations in both hereditary and sporadic MTC and PC development are largely unknown. Here, we show for the first time the presence of somatic mutations in the cell cycle regulator P18 in human RET-associated MTCs and PCs. Each of these mutations causes an amino acid substitution in the cyclin dependent kinase-interacting region of P18(INK4C). Since these mutations partly inhibited P18(INK4C) function and reduced its stability, our findings implicate P18 as a tumor suppressor gene involved in human MTC and PC development.