Wnt/PCP signaling mediates breast cancer metastasis by promoting pro-invasive protrusion formation in collectively motile leader cells

As evidence supporting essential roles for collective cell migration in carcinoma metastasis continues to accumulate, a better understanding of the underlying cellular and molecular mechanisms will be critical to translating these findings to the treatment of advanced cancers. Here we report that Wnt/PCP, a non-canonical Wnt signaling pathway, mediates breast cancer collective migration and metastasis. We observe that mammary gland-specific knockout of Vangl2, a tetraspanin-like scaffolding protein required for Wnt5a-induced signaling and motility in cultured breast cancer cell lines, results in a striking decrease in metastatic efficiency but not primary tumor growth in the MMTV-NDL transgenic mouse model of HER2-positive breast cancer. We also observe that expression levels of core Wnt/PCP components Wnt5a, Vangl1 and Vangl2 are selectively elevated in K14-positive leader cells relative to follower cells within a collectively migrating cohort, and that Vangl2 expression selectively promotes RhoA activation in leading edge cells. Moreover, Vangl expression drives collective migration in three-dimensional ex vivo tumor organoids, and Vangl protein specifically accumulates within pro-migratory filamentous actin-rich protrusions of leader cells. Together, our observations point to a model whereby Wnt/PCP upregulation facilitates breast tumor collective cell motility by selectively augmenting the formation pro-migratory protrusions within leader cells.

Tracking of Endothelial Cell Migration and Stiffness Measurements Reveal the Role of Cytoskeletal Dynamics

Cell migration is a complex, tightly regulated multistep process in which cytoskeletal reorganization and focal adhesion redistribution play a central role. Core to both individual and collective migration is the persistent random walk, which is characterized by random force generation and resistance to directional change. We first discuss a model that describes the stochastic movement of ECs and characterizes EC persistence in wound healing. To that end, we pharmacologically disrupted cytoskeletal dynamics, cytochalasin D for actin and nocodazole for tubulin, to understand its contributions to cell morphology, stiffness, and motility. As such, the use of Atomic Force Microscopy (AFM) enabled us to probe the topography and stiffness of ECs, while time lapse microscopy provided observations in wound healing models. Our results suggest that actin and tubulin dynamics contribute to EC shape, compressive moduli, and directional organization in collective migration. Insights from the model and time lapse experiment suggest that EC speed and persistence are directionally organized in wound healing. Pharmacological disruptions suggest that actin and tubulin dynamics play a role in collective migration. Current insights from both the model and experiment represent an important step in understanding the biomechanics of EC migration as a therapeutic target.

Nuclear lamins promote protrusion dynamics and collective, confined migration in vivo

Cells migrate collectively through confined environments during development and cancer metastasis. While the nucleus, a large and stiff organelle, impedes cell migration between non-deformable pillars in vitro, its function in vivo may vary depending on the microenvironment. Further, it is unknown how nuclei contribute to collective migration in vivo and whether nuclei in different positions within cell collectives experience different forces. Here, we use border cell migration in the fly ovary as an in vivo model to investigate the effects of confined, collective migration on nuclei and the contribution of nuclear lamins to migration. We found severe yet transient nuclear deformations occur, particularly in the leading cell, as border cells squeeze through tiny crevices between germline cells, termed nurse cells. Leading cells extend protrusions between nurse cells, which may pry open space to allow the cluster to advance. Here we report that the leading cell nuclei deformed as they moved into leading protrusions. Then as protrusions widened, the nucleus recovered a more circular shape. These data suggest that lead cell nuclei may help protrusions expand and thereby enlarge the migration path. To test how nuclei might promote or impede border cell migration, we investigated nuclear lamins, proteins that assemble into intermediate filaments and structurally support the nuclear envelope. Depletion of the Drosophila B-type lamin, Lam, from the outer, motile border cells, but not the inner, nonmotile polar cells, impeded border cell migration, whereas perturbations of the A-type lamin, LamC, did not. While wild type border cell clusters typically have one large leading protrusion as they delaminate from the anterior follicular epithelium, clusters depleted of B-type lamin had multiple, short-lived protrusions, resulting in unproductive cluster movement and failure to progress along the migration path. Further, border cell nuclei depleted of B-type lamins were small, formed blebs, and ruptured. Together, these data indicate that B-type lamin is requied for nuclear integrity, which in turn stabilizes the leading protrusion and promotes overall cluster polarization and collective movement through confined spaces.

PTEN inhibits AMPK to control collective migration

PTEN is one of the most frequently mutated tumor suppressor gene in cancer. PTEN is generally altered in invasive cancers such as glioblastomas, but its function in collective cell migration and invasion is not fully characterized. Herein, we report that the loss of PTEN increases cell speed during collective migration of non-tumourous cells both in vitro and in vivo. We further show that loss of PTEN promotes LKB1-dependent phosphorylation and activation of the major metabolic regulator AMPK. In turn AMPK increases VASP phosphorylation, reduces VASP localization at cell-cell junctions and decreases the interjunctional transverse actin arcs at the leading front, provoking a weakening of cell-cell contacts and increasing migration speed. Targeting AMPK activity not only slows down PTEN-depleted cells, it also limits PTEN-null glioblastoma cell invasion, opening new opportunities to treat glioblastoma lethal invasiveness.

Spatiotemporal dynamics of PIEZO1 localization controls keratinocyte migration during wound healing

Keratinocytes, the predominant cell type of the epidermis, migrate to reinstate the epithelial barrier during wound healing. Mechanical cues are known to regulate keratinocyte re-epithelialization and wound healing however, the underlying molecular transducers and biophysical mechanisms remain elusive. Here, we show through molecular, cellular and organismal studies that the mechanically-activated ion channel PIEZO1 regulates keratinocyte migration and wound healing. Epidermal-specific Piezo1 knockout mice exhibited faster wound closure while gain-of-function mice displayed slower wound closure compared to littermate controls. By imaging the spatiotemporal localization dynamics of endogenous PIEZO1 channels we find that channel enrichment at some regions of the wound edge induces a localized cellular retraction that slows keratinocyte collective migration. In migrating single keratinocytes, PIEZO1 is enriched at the rear of the cell, where maximal retraction occurs, and we find that chemical activation of PIEZO1 enhances retraction during single as well as collective migration. Our findings uncover novel molecular mechanisms underlying single and collective keratinocyte migration that may suggest a potential pharmacological target for wound treatment. More broadly, we show that nanoscale spatiotemporal dynamics of Piezo1 channels can control tissue-scale events, a finding with implications beyond wound healing to processes as diverse as development, homeostasis, disease and repair.

Fast transcriptional activation of developmental signalling pathways during wound healing of the calcareous sponge Sycon ciliatum

Background: The ability to regenerate lost or damaged body parts is an ancient animal characteristic with a wide yet variable distribution across all phyla. Sponges, likely the sister group to all other animals, have remarkable regenerative abilities including whole body regeneration and re-development from dissociated cells. The calcareous sponge Sycon ciliatum has been subject to various regeneration studies since the beginning of the last century. However, the early steps of wound healing of S. ciliatum have not been addressed from the molecular perspective. Results: In this study, we combined electron microscopy with gene expression analysis to investigate wound healing after transverse sectioning of S. ciliatum. Microscopic analysis revealed massive transdifferentiation and collective migration behaviour of choanocytes and pinacocytes early upon injury (6-12h) as the main mechanisms for quick closure of the wound surface. RNA-sequencing identified upregulation of components of the conserved metazoan Wnt and TGFβ signalling pathways within 3h, preceding morphologically detectable wound healing events. De novo upregulation after a decline in expression coincides with morphologically visible polarity establishment. Moreover, by integrating the new wound healing data set with previously published data derived from intact sponge, we demonstrate similarity between gene activity during early wound healing and osculum maintenance. Whole mount in situ hybridisation of the TGFβ signalling pathway ligand SciTGFβU and signal transducer SciSmadRa show that the early activation of both is initially encompassing a large area surrounding the cut surface with gradual restriction to the edge of the forming regenerative membrane as wound healing progresses. While SciTGFβU transcripts are localised to exo- and endopinacocytes, SciSmadRa expression appears across all cell types. Using an EdU cell proliferation assay, we found that a global increase in cell proliferation is not visible before 12h into wound healing. Hence, the initial stages to cover the injury site including cell transdifferentiation and migration seem to be executed by cells remaining after injury. Gene expression clustering coupled with GO term enrichment analysis confirmed that expression of genes involved in processes related to cell proliferation, DNA repair as well as apoptotic processes at 3 and 6h of wound healing was not upregulated. On the other hand, genes associated with positive regulation of transcription, signal transduction, actin filament and chromatin organisation, as well as the Wnt signalling pathway are upregulated at early wound healing stages. Conclusion: We have analysed wound healing in the calcareous sponge Sycon ciliatum using microscopic and genomic methods. This study highlights a remarkable mechanism of interplay between cell transdifferentiation and collective migration we hypothesise to be regulated by conserved metazoan developmental pathways and numerous taxonomically restricted genes. Expression of these genes in regenerating and intact sponges sheds light on the long-standing question whether embryonic developmental pathways are redeployed in regeneration.