Integrin Mechano-chemical Signaling Generates Plasma Membrane Nanodomains that Promote Cell Spreading.

Glycosylphosphatidylinositol-anchored proteins (GPI-APs) are a major class of lipid-anchored plasma membrane proteins. GPI-APs form nanoclusters generated by cortical acto-myosin activity. While our understanding of the physical principles governing this process is emerging, the molecular machinery and functional relevance of GPI-AP nanoclustering are unknown. Here, we first show that a membrane receptor signaling pathway directs nanocluster formation. Arg-Gly-Asp motif-containing ligands bound to the ß1-integrin receptor activate src and focal adhesion kinases, resulting in RhoA signaling. This cascade triggers actin-nucleation via specific formins, which, along with myosin activity, drive the nanoclustering of membrane proteins with actin-binding domains. Concurrently, talin-mediated activation of the mechano-transducer vinculin is required for the coupling of the acto-myosin machinery to inner-leaflet lipids, thereby generating GPI-AP nanoclusters. Second, we show that these nanoclusters are functional; disruption of their formation either in GPI-anchor remodeling mutants or in vinculin mutants impairs cell spreading and migration, hallmarks of integrin function.

Cytokinesis failure triggers hippo tumor suppressor pathway activation.

Genetically unstable tetraploid cells can promote tumorigenesis. Recent estimates suggest that ∼37% of human tumors have undergone a genome-doubling event during their development. This potentially oncogenic effect of tetraploidy is countered by a p53-dependent barrier to proliferation. However, the cellular defects and corresponding signaling pathways that trigger growth suppression in tetraploid cells are not known. Here, we combine RNAi screening and in vitro evolution approaches to demonstrate that cytokinesis failure activates the Hippo tumor suppressor pathway in cultured cells, as well as in naturally occurring tetraploid cells in vivo. Induction of the Hippo pathway is triggered in part by extra centrosomes, which alter small G protein signaling and activate LATS2 kinase. LATS2 in turn stabilizes p53 and inhibits the transcriptional regulators YAP and TAZ. These findings define an important tumor suppression mechanism and uncover adaptive mechanisms potentially available to nascent tumor cells that bypass this inhibitory regulation.

Fever Promotes T Lymphocyte Trafficking via a Thermal Sensory Pathway Involving Heat Shock Protein 90 and α4 Integrins.

Fever is an evolutionarily conserved response that confers survival benefits during infection. However, the underlying mechanism remains obscure. Here, we report that fever promoted T lymphocyte trafficking through heat shock protein 90 (Hsp90)-induced α4 integrin activation and signaling in T cells. By inducing selective binding of Hsp90 to α4 integrins, but not ß2 integrins, fever increased α4-integrin-mediated T cell adhesion and transmigration. Mechanistically, Hsp90 bound to the α4 tail and activated α4 integrins via inside-out signaling. Moreover, the N and C termini of one Hsp90 molecule simultaneously bound to two α4 tails, leading to dimerization and clustering of α4 integrins on the cell membrane and subsequent activation of the FAK-RhoA pathway. Abolishment of Hsp90-α4 interaction inhibited fever-induced T cell trafficking to draining lymph nodes and impaired the clearance of bacterial infection. Our findings identify the Hsp90-α4-integrin axis as a thermal sensory pathway that promotes T lymphocyte trafficking and enhances immune surveillance during infection.

Substrate stress relaxation regulates neural stem cell fate commitment.

Adult neural stem cells (NSCs) reside in the dentate gyrus of the hippocampus, and their capacity to generate neurons and glia plays a role in learning and memory. In addition, neurodegenerative diseases are known to be caused by a loss of neurons and glial cells, resulting in a need to better understand stem cell fate commitment processes. We previously showed that NSC fate commitment toward a neuronal or glial lineage is strongly influenced by extracellular matrix stiffness, a property of elastic materials. However, tissues in vivo are not purely elastic and have varying degrees of viscous character. Relatively little is known about how the viscoelastic properties of the substrate impact NSC fate commitment. Here, we introduce a polyacrylamide-based cell culture platform that incorporates mismatched DNA oligonucleotide-based cross-links as well as covalent cross-links. This platform allows for tunable viscous stress relaxation properties via variation in the number of mismatched base pairs. We find that NSCs exhibit increased astrocytic differentiation as the degree of stress relaxation is increased. Furthermore, culturing NSCs on increasingly stress-relaxing substrates impacts cytoskeletal dynamics by decreasing intracellular actin flow rates and stimulating cyclic activation of the mechanosensitive protein RhoA. Additionally, inhibition of motor-clutch model components such as myosin II and focal adhesion kinase partially or completely reverts cells to lineage distributions observed on elastic substrates. Collectively, our results introduce a unique system for controlling matrix stress relaxation properties and offer insight into how NSCs integrate viscoelastic cues to direct fate commitment.

Hydrogel biomaterials that stiffen and soften on demand reveal that skeletal muscle stem cells harbor a mechanical memory.

Muscle stem cells (MuSCs) are specialized cells that reside in adult skeletal muscle poised to repair muscle tissue. The ability of MuSCs to regenerate damaged tissues declines markedly with aging and in diseases such as Duchenne muscular dystrophy, but the underlying causes of MuSC dysfunction remain poorly understood. Both aging and disease result in dramatic increases in the stiffness of the muscle tissue microenvironment from fibrosis. MuSCs are known to lose their regenerative potential if cultured on stiff plastic substrates. We sought to determine whether MuSCs harbor a memory of their past microenvironment and if it can be overcome. We tested MuSCs in situ using dynamic hydrogel biomaterials that soften or stiffen on demand in response to light and found that freshly isolated MuSCs develop a persistent memory of substrate stiffness characterized by loss of proliferative progenitors within the first three days of culture on stiff substrates. MuSCs cultured on soft hydrogels had altered cytoskeletal organization and activity of Rho and Rac guanosine triphosphate hydrolase (GTPase) and Yes-associated protein mechanotransduction pathways compared to those on stiff hydrogels. Pharmacologic inhibition identified RhoA activation as responsible for the mechanical memory phenotype, and single-cell RNA sequencing revealed a molecular signature of the mechanical memory. These studies highlight that microenvironmental stiffness regulates MuSC fate and leads to MuSC dysfunction that is not readily reversed by changing stiffness. Our results suggest that stiffness can be circumvented by targeting downstream signaling pathways to overcome stem cell dysfunction in aged and disease states with aberrant fibrotic tissue mechanics.

Dual spatio-temporal regulation of axon growth and microtubule dynamics by RhoA signaling pathways.

RhoA plays a crucial role in neuronal polarization, where its action restraining axon outgrowth has been thoroughly studied. We now report that RhoA has not only an inhibitory but also a stimulatory effect on axon development depending on when and where exerts its action and the downstream effectors involved. In cultured hippocampal neurons, FRET imaging revealed that RhoA activity selectively localized in growth cones of undifferentiated neurites, whereas in developing axons it displayed a biphasic pattern, being low in nascent axons and high in elongating ones. RhoA-Rho kinase (ROCK) signaling prevented axon initiation but had no effect on elongation, whereas formin inhibition reduced axon extension without significantly altering initial outgrowth. In addition, RhoA-mDia signaling promoted axon elongation by stimulating growth cone microtubule stability and assembly, as opposed to RhoA-ROCK signaling, which restrained growth cone microtubule assembly and protrusion.

Mechanosensitive stem cell fate choice is instructed by dynamic fluctuations in activation of Rho GTPases.

During the intricate process by which cells give rise to tissues, embryonic and adult stem cells are exposed to diverse mechanical signals from the extracellular matrix (ECM) that influence their fate. Cells can sense these cues in part through dynamic generation of protrusions, modulated and controlled by cyclic activation of Rho GTPases. However, it remains unclear how extracellular mechanical signals regulate Rho GTPase activation dynamics and how such rapid, transient activation dynamics are integrated to yield long-term, irreversible cell fate decisions. Here, we report that ECM stiffness cues alter not only the magnitude but also the temporal frequency of RhoA and Cdc42 activation in adult neural stem cells (NSCs). Using optogenetics to control the frequency of RhoA and Cdc42 activation, we further demonstrate that these dynamics are functionally significant, where high- vs. low-frequency activation of RhoA and Cdc42 drives astrocytic vs. neuronal differentiation, respectively. In addition, high-frequency Rho GTPase activation induces sustained phosphorylation of the TGFß pathway effector SMAD1, which in turn drives the astrocytic differentiation. By contrast, under low-frequency Rho GTPase stimulation, cells fail to accumulate SMAD1 phosphorylation and instead undergo neurogenesis. Our findings reveal the temporal patterning of Rho GTPase signaling and the resulting accumulation of an SMAD1 signal as a critical mechanism through which ECM stiffness cues regulate NSC fate.

The small GTPase Cdc42 regulates shell field morphogenesis in a gastropod mollusk.

In most mollusks (conchiferans), the early tissue responsible for shell development, namely, the shell field, shows a common process of invagination during morphogenesis. Moreover, lines of evidence indicated that shell field invagination is not an independent event, but an integrated output reflecting the overall state of shell field morphogenesis. Nevertheless, the underlying mechanisms of this conserved process remain largely unknown. We previously found that actomyosin networks (regularly organized filamentous actin (F-actin) and myosin) may play essential roles in this process by revealing the evident aggregation of F-actin in the invaginated region and demonstrating that nonmuscle myosin II (NM II) is required for invagination in the gastropod Lottia peitaihoensis (= Lottia goshimai). Here, we investigated the roles of the Rho family of small GTPases (RhoA, Rac1, and Cdc42) to explore the upstream regulators of actomyosin networks. Functional assays using small molecule inhibitors suggested that Cdc42 modulates key events of shell field morphogenesis, including invagination and cell rearrangements, while the roles of RhoA and Rac1 may be nonspecific or negligible. Further investigations revealed that the Cdc42 protein was concentrated on the apical side of shell field cells and colocalized with F-actin aggregation. The aggregation of these two molecules could be prevented by treatment with Cdc42 inhibitors. These findings suggest a possible regulatory cascade of shell field morphogenesis in which Cdc42 recruits F-actin (actomyosin networks) on the apical side of shell field cells, which then generates resultant mechanical forces that mediate correct shell field morphogenesis (cell shape changes, invagination and cell rearrangement). Our results emphasize the roles of the cytoskeleton in early shell development and provide new insights into molluscan shell evolution.

Tyrosine-modifying glycosylation by Yersinia effectors.

Mono-O-glycosylation of target proteins by bacterial toxins or effector proteins is a well-known mechanism by which bacteria interfere with essential functions of host cells. The respective glycosyltransferases are important virulence factors such as the Clostridioides difficile toxins A and B. Here, we describe two glycosyltransferases of Yersinia species that have a high sequence identity: YeGT from the zoonotic pathogen Yersinia enterocolitica and YkGT from the murine pathogen Yersinia kristensenii. We show that both modify Rho family proteins by attachment of GlcNAc at tyrosine residues (Tyr-34 in RhoA). Notably, the enzymes differed in their target protein specificity. While YeGT modified RhoA, B, and C, YkGT possessed a broader substrate spectrum and glycosylated not only Rho but also Rac and Cdc42 subfamily proteins. Mutagenesis studies indicated that residue 177 is important for this broader target spectrum. We determined the crystal structure of YeGT shortened by 16 residues N terminally (sYeGT) in the ligand-free state and bound to UDP, the product of substrate hydrolysis. The structure assigns sYeGT to the GT-A family. It shares high structural similarity to glycosyltransferase domains from toxins. We also demonstrated that the 16 most N-terminal residues of YeGT and YkGT are important for the mediated translocation into the host cell using the pore-forming protective antigen of anthrax toxin. Mediated introduction into HeLa cells or ectopic expression of YeGT and YkGT caused morphological changes and redistribution of the actin cytoskeleton. The data suggest that YeGT and YkGT are likely bacterial effectors belonging to the family of tyrosine glycosylating bacterial glycosyltransferases.

Interplay of RAP2 GTPase and the cytoskeleton in Hippo pathway regulation.

The Hippo signaling is instrumental in regulating organ size, regeneration, and carcinogenesis. The cytoskeleton emerges as a primary Hippo signaling modulator. Its structural alterations in response to environmental and intrinsic stimuli control Hippo signaling pathway activity. However, the precise mechanisms underlying the cytoskeleton regulation of Hippo signaling are not fully understood. RAP2 GTPase is known to mediate the mechanoresponses of Hippo signaling via activating the core Hippo kinases LATS1/2 through MAP4Ks and MST1/2. Here we show the pivotal role of the reciprocal regulation between RAP2 GTPase and the cytoskeleton in Hippo signaling. RAP2 deletion undermines the responses of the Hippo pathway to external cues tied to RhoA GTPase inhibition and actin cytoskeleton remodeling, such as energy stress and serum deprivation. Notably, RhoA inhibitors and actin disruptors fail to activate LATS1/2 effectively in RAP2-deficient cells. RNA sequencing highlighted differential regulation of both actin and microtubule networks by RAP2 gene deletion. Consistently, Taxol, a microtubule-stabilizing agent, was less effective in activating LATS1/2 and inhibiting cell growth in RAP2 and MAP4K4/6/7 knockout cells. In summary, our findings position RAP2 as a central integrator of cytoskeletal signals for Hippo signaling, which offers new avenues for understanding Hippo regulation and therapeutic interventions in Hippo-impaired cancers.

Crystal structure of bacterial cytotoxic necrotizing factor CNFY reveals molecular building blocks for intoxication.

Cytotoxic necrotizing factors (CNFs) are bacterial single-chain exotoxins that modulate cytokinetic/oncogenic and inflammatory processes through activation of host cell Rho GTPases. To achieve this, they are secreted, bind surface receptors to induce endocytosis and translocate a catalytic unit into the cytosol to intoxicate host cells. A three-dimensional structure that provides insight into the underlying mechanisms is still lacking. Here, we determined the crystal structure of full-length Yersinia pseudotuberculosis CNFY . CNFY consists of five domains (D1-D5), and by integrating structural and functional data, we demonstrate that D1-3 act as export and translocation module for the catalytic unit (D4-5) and for a fused ß-lactamase reporter protein. We further found that D4, which possesses structural similarity to ADP-ribosyl transferases, but had no equivalent catalytic activity, changed its position to interact extensively with D5 in the crystal structure of the free D4-5 fragment. This liberates D5 from a semi-blocked conformation in full-length CNFY , leading to higher deamidation activity. Finally, we identify CNF translocation modules in several uncharacterized fusion proteins, which suggests their usability as a broad-specificity protein delivery tool.

RASSF1C oncogene elicits amoeboid invasion, cancer stemness, and extracellular vesicle release via a SRC/Rho axis.

Cell plasticity is a crucial hallmark leading to cancer metastasis. Upregulation of Rho/ROCK pathway drives actomyosin contractility, protrusive forces, and contributes to the occurrence of highly invasive amoeboid cells in tumors. Cancer stem cells are similarly associated with metastasis, but how these populations arise in tumors is not fully understood. Here, we show that the novel oncogene RASSF1C drives mesenchymal-to-amoeboid transition and stem cell attributes in breast cancer cells. Mechanistically, RASSF1C activates Rho/ROCK via SRC-mediated RhoGDI inhibition, resulting in generation of actomyosin contractility. Moreover, we demonstrate that RASSF1C-induced amoeboid cells display increased expression of cancer stem-like markers such as CD133, ALDH1, and Nanog, and are accompanied by higher invasive potential in vitro and in vivo. Further, RASSF1C-induced amoeboid cells employ extracellular vesicles to transfer the invasive phenotype to target cells and tissue. Importantly, the underlying RASSF1C-driven biological processes concur to explain clinical data: namely, methylation of the RASSF1C promoter correlates with better survival in early-stage breast cancer patients. Therefore, we propose the use of RASSF1 gene promoter methylation status as a biomarker for patient stratification.

Lfc subcellular localization and activity is controlled by αv-class integrin.

Fibronectin (FN)-binding integrins control a variety of cellular responses through Rho GTPases. The FN-binding integrins, αvß3 and α5ß1, are known to induce different effects on cell morphology and motility. Here, we report that FN-bound αvß3 integrin, but not FN-bound α5ß1 integrin, triggers the dissociation of the RhoA GEF Lfc (also known as GEF-H1 and ARHGEF2 in humans) from microtubules (MTs), leading to the activation of RhoA, formation of stress fibres and maturation of focal adhesions (FAs). Conversely, loss of Lfc expression decreases RhoA activity, stress fibre formation and FA size, suggesting that Lfc is the major GEF downstream of FN-bound αvß3 that controls RhoA activity. Mechanistically, FN-engaged αvß3 integrin activates a kinase cascade involving MARK2 and MARK3, which in turn leads to phosphorylation of several phospho-sites on Lfc. In particular, S151 was identified as the main site involved in the regulation of Lfc localization and activity. Our findings indicate that activation of Lfc and RhoA is orchestrated in FN-adherent cells in an integrin-specific manner.

Co-culture of RhoA-overexpressed microtia chondrocytes and adipose-derived stem cells in the construction of tissue-engineered ear-shaped cartilage.

Microtia is a congenital auricle dysplasia with a high incidence and tissue engineering technology provides a promising strategy to reconstruct auricles. We previously described that the engineered cartilage constructed from microtia chondrocytes exhibited inferior levels of biochemical and biomechanical properties, which was proposed to be resulted of the decreased migration ability of microtia chondrocytes. In the current study, we found that Rho GTPase members were deficient in microtia chondrocytes. By overexpressing RhoA, Rac1, and CDC42, respectively, we further demonstrated that RhoA took great responsibility for the decreased migration ability of microtia chondrocytes. Moreover, we constructed PGA/PLA scaffold-based cartilages to verify the chondrogenic ability of RhoA overexpressed microtia chondrocytes, and the results showed that overexpressing RhoA was of limited help in improving the quality of microtia chondrocyte engineered cartilage. However, coculture of adipose-derived stem cells (ADSCs) significantly improved the biochemical and biomechanical properties of engineered cartilage. Especially, coculture of RhoA overexpressed microtia chondrocytes and ADSCs produced an excellent effect on the wet weight, cartilage-specific extracellular matrix, and biomechanical property of engineered cartilage. Furthermore, we presented that coculture of RhoA overexpressed microtia chondrocytes and ADSCs combined with human ear-shaped PGA/PLA scaffold and titanium alloy stent fabricated by CAD/CAM and 3D printing technology effectively constructed and maintained auricle structure in vivo. Collectively, our results provide evidence for the essential role of RhoA in microtia chondrocytes and a developed strategy for the construction of patient-specific tissue-engineered auricular cartilage.

Molecular mechanism of bitter taste receptor agonist-mediated relaxation of airway smooth muscle.

G-protein-coupled receptors (GPCRs) belonging to the type 2 taste receptors (TAS2Rs) family are predominantly present in taste cells to allow the perception of bitter-tasting compounds. TAS2Rs have also been shown to be expressed in human airway smooth muscle (ASM), and TAS2R agonists relax ASM cells and bronchodilate airways despite elevating intracellular calcium. This calcium "paradox" (calcium mediates contraction by pro-contractile Gq-coupled GPCRs) and the mechanisms by which TAS2R agonists relax ASM remain poorly understood. To gain insight into pro-relaxant mechanisms effected by TAS2Rs, we employed an unbiased phosphoproteomic approach involving dual-mass spectrometry to determine differences in the phosphorylation of contractile-related proteins in ASM following the stimulation of cells with TAS2R agonists, histamine (an agonist of the Gq-coupled H1 histamine receptor) or isoproterenol (an agonist of the Gs-coupled ß2-adrenoceptor) alone or in combination. Our study identified differential phosphorylation of proteins regulating contraction, including A-kinase anchoring protein (AKAP)2, AKAP12, and RhoA guanine nucleotide exchange factor (ARHGEF)12. Subsequent signaling analyses revealed RhoA and the T853 residue on myosin light chain phosphatase (MYPT)1 as points of mechanistic divergence between TAS2R and Gs-coupled GPCR pathways. Unlike Gs-coupled receptor signaling, which inhibits histamine-induced myosin light chain (MLC)20 phosphorylation via protein kinase A (PKA)-dependent inhibition of intracellular calcium mobilization, HSP20 and ERK1/2 activity, TAS2Rs are shown to inhibit histamine-induced pMLC20 via inhibition of RhoA activity and MYPT1 phosphorylation at the T853 residue. These findings provide insight into the TAS2R signaling in ASM by defining a distinct signaling mechanism modulating inhibition of pMLC20 to relax contracted ASM.

EphB-mediated degradation of the RhoA GEF Ephexin5 relieves a developmental brake on excitatory synapse formation.

The mechanisms that promote excitatory synapse formation and maturation have been extensively studied. However, the molecular events that limit excitatory synapse development so that synapses form at the right time and place and in the correct numbers are less well understood. We have identified a RhoA guanine nucleotide exchange factor, Ephexin5, which negatively regulates excitatory synapse development until EphrinB binding to the EphB receptor tyrosine kinase triggers Ephexin5 phosphorylation, ubiquitination, and degradation. The degradation of Ephexin5 promotes EphB-dependent excitatory synapse development and is mediated by Ube3A, a ubiquitin ligase that is mutated in the human cognitive disorder Angelman syndrome and duplicated in some forms of Autism Spectrum Disorders (ASDs). These findings suggest that aberrant EphB/Ephexin5 signaling during the development of synapses may contribute to the abnormal cognitive function that occurs in Angelman syndrome and, possibly, ASDs.

Structural basis for the distinct roles of non-conserved Pro116 and conserved Tyr124 of BCH domain of yeast p50RhoGAP.

p50RhoGAP is a key protein that interacts with and downregulates the small GTPase RhoA. p50RhoGAP is a multifunctional protein containing the BNIP-2 and Cdc42GAP Homology (BCH) domain that facilitates protein-protein interactions and lipid binding and the GAP domain that regulates active RhoA population. We recently solved the structure of the BCH domain from yeast p50RhoGAP (YBCH) and showed that it maintains the adjacent GAP domain in an auto-inhibited state through the ß5 strand. Our previous WT YBCH structure shows that a unique kink at position 116 thought to be made by a proline residue between alpha helices α6 and α7 is essential for the formation of intertwined dimer from asymmetric monomers. Here we sought to establish the role and impact of this Pro116. However, the kink persists in the structure of P116A mutant YBCH domain, suggesting that the scaffold is not dictated by the proline residue at this position. We further identified Tyr124 (or Tyr188 in HBCH) as a conserved residue in the crucial ß5 strand. Extending to the human ortholog, when substituted to acidic residues, Tyr188D or Tyr188E, we observed an increase in RhoA binding and self-dimerization, indicative of a loss of inhibition of the GAP domain by the BCH domain. These results point to distinct roles and impact of the non-conserved and conserved amino acid positions in regulating the structural and functional complexity of the BCH domain.

Plakophilin 4 controls the spatio-temporal activity of RhoA at adherens junctions to promote cortical actin ring formation and tissue tension.

Plakophilin 4 (PKP4) is a component of cell-cell junctions that regulates intercellular adhesion and Rho-signaling during cytokinesis with an unknown function during epidermal differentiation. Here we show that keratinocytes lacking PKP4 fail to develop a cortical actin ring, preventing adherens junction maturation and generation of tissue tension. Instead, PKP4-depleted cells display increased stress fibers. PKP4-dependent RhoA localization at AJs was required to activate a RhoA-ROCK2-MLCK-MLC2 axis and organize actin into a cortical ring. AJ-associated PKP4 provided a scaffold for the Rho activator ARHGEF2 and the RhoA effectors MLCK and MLC2, facilitating the spatio-temporal activation of RhoA signaling at cell junctions to allow cortical ring formation and actomyosin contraction. In contrast, association of PKP4 with the Rho suppressor ARHGAP23 reduced ARHGAP23 binding to RhoA which prevented RhoA activation in the cytoplasm and stress fiber formation. These data identify PKP4 as an AJ component that transduces mechanical signals into cytoskeletal organization.

Spindle reorientation in response to mechanical stress is an emergent property of the spindle positioning mechanisms.

Proper orientation of the mitotic spindle plays a crucial role in embryos, during tissue development, and in adults, where it functions to dissipate mechanical stress to maintain tissue integrity and homeostasis. While mitotic spindles have been shown to reorient in response to external mechanical stresses, the subcellular cues that mediate spindle reorientation remain unclear. Here, we used a combination of optogenetics and computational modeling to investigate how mitotic spindles respond to inhomogeneous tension within the actomyosin cortex. Strikingly, we found that the optogenetic activation of RhoA only influences spindle orientation when it is induced at both poles of the cell. Under these conditions, the sudden local increase in cortical tension induced by RhoA activation reduces pulling forces exerted by cortical regulators on astral microtubules. This leads to a perturbation of the balance of torques exerted on the spindle, which causes it to rotate. Thus, spindle rotation in response to mechanical stress is an emergent phenomenon arising from the interaction between the spindle positioning machinery and the cell cortex.

Progressive polarity loss and luminal collapse disrupt tissue organization in carcinoma.

Epithelial cancers (carcinoma) account for 80%-90% of all cancers. The development of carcinoma is associated with disrupted epithelial organization and solid ductal structures. The mechanisms underlying the morphological development of carcinoma are poorly understood, but it is thought that loss of cell polarity is an early event. Here we report the characterization of the development of human breast lesions leading to carcinoma. We identified a unique mechanism that generates solid ducts in carcinoma through progressive loss of polarity and collapse of the luminal architecture. This program initiates with asymmetric divisions of polarized cells that generate a stratified epithelium containing both polarized and depolarized cells. Stratified regions form cords that penetrate into the lumen, subdividing it into polarized secondary lumina. The secondary lumina then collapse with a concomitant decrease in RhoA and myosin II activity at the apical membrane and ultimately lose apical-basal polarity. By restoring RhoA activity in mice, ducts maintained lumen and cell polarity. Notably, disrupted tissue architecture through luminal collapse was reversible, and ducts with a lumen were re-established after oncogene suppression in vivo. This reveals a novel and common mechanism that contributes to carcinoma development by progressively disrupting cell and tissue organization.