EMT, the cytoskeleton, and cancer cell invasion

The metastatic process, i.e. the dissemination of cancer cells throughout the body to seed secondary tumors at distant sites, requires cancer cells to leave the primary tumor and to acquire migratory and invasive capabilities. In a process of epithelial-mesenchymal transition (EMT), besides changing their adhesive repertoire, cancer cells employ developmental processes to gain migratory and invasive properties that involve a dramatic reorganization of the actin cytoskeleton and the concomitant formation of membrane protrusions required for invasive growth. The molecular processes underlying such cellular changes are still only poorly understood, and the various migratory organelles, including lamellipodia, filopodia, invadopodia and podosomes, still require a better functional and molecular characterization. Notably, direct experimental evidence linking the formation of migratory membrane protrusions and the process of EMT and tumor metastasis is still lacking. In this review, we have summarized recent novel insights into the molecular processes and players underlying EMT on one side and the formation of invasive membrane protrusions on the other sid

Sprouty proteins, masterminds of receptor tyrosine kinase signaling

Angiogenesis relies on endothelial cells properly processing signals from growth factors provided in both an autocrine and a paracrine manner. These mitogens bind to their cognate receptor tyrosine kinases (RTKs) on the cell surface, thereby activating a myriad of complex intracellular signaling pathways whose outputs include cell growth, migration, and morphogenesis. Understanding how these cascades are precisely controlled will provide insight into physiological and pathological angiogenesis. The Sprouty (Spry) family of proteins is a highly conserved group of negative feedback loop modulators of growth factor-mediated mitogen-activated protein kinase (MAPK) activation originally described in Drosophila. There are four mammalian orthologs (Spry1-4) whose modulation of RTK-induced signaling pathways is growth factor- and cell context-dependant. Endothelial cells are a group of highly differentiated cell types necessary for defining the mammalian vasculature. These cells respond to a plethora of growth factors and express all four Spry isoforms, thus highlighting the complexity that is required to form and maintain vessels in mammals. This review describes Spry functions in the context of endothelial biology and angiogenesis, and provides an update on Spry-interacting proteins and Spry mechanisms of actio

Heterogeneity for IGF-II production maintained by public goods dynamics in neuroendocrine pancreatic cancer

The extensive intratumor heterogeneity revealed by sequencing cancer genomes is an essential determinant of tumor progression, diagnosis, and treatment. What maintains heterogeneity remains an open question because competition within a tumor leads to a strong selection for the fittest subclone. Cancer cells also cooperate by sharing molecules with paracrine effects, such as growth factors, and heterogeneity can be maintained if subclones depend on each other for survival. Without strict interdependence between subclones, however, nonproducer cells can free-ride on the growth factors produced by neighboring producer cells, a collective action problem known in game theory as the “tragedy of the commons,” which has been observed in microbial cell populations. Here, we report that similar dynamics occur in cancer cell populations. Neuroendocrine pancreatic cancer (insulinoma) cells that do not produce insulin-like growth factor II (IGF-II) grow slowly in pure cultures but have a proliferation advantage in mixed cultures, where they can use the IGF-II provided by producer cells. We show that, as predicted by evolutionary game theory, producer cells do not go extinct because IGF-II acts as a nonlinear public good, creating negative frequency-dependent selection that leads to a stable coexistence of the two cell types. Intratumor cell heterogeneity can therefore be maintained even without strict interdependence between cell subclones. Reducing the amount of growth factors available within a tumor may lead to a reduction in growth followed by a new equilibrium, which may explain relapse in therapies that target growth factors

Reciprocal regulatory circuits coordinating EMT plasticity

Epithelial to mesenchymal transition (EMT) as well as its reversal process, mesenchymal to epithelial transition (MET), are essential and well-controlled cellular processes during embryonic development. Tightly controlled regulatory mechanisms guide an EMT/MET plasticity and enable cells to switch forth and back between different cell morphologies and functional capabilities to endow the necessity of tissue plasticity. However, aberrant and uncontrolled activation of these processes during malignant tumor progression promotes primary tumor cell invasion, cancer cell dissemination and metastatic outgrowth. In a recent study (Nat Commun; doi: 10.1038/s41467-017-01197-w), we have reported on the post-transcriptional control of normal and cancer-associated EMT by miRNAs and identified a novel, critical double-negative feedback regulation of the thus far unknown miRNA miR1199 and the key EMT transcription factor Zeb1

Sprouty2 expression controls endothelial monolayer integrity and quiescence

Vascular integrity is fundamental to the formation of mature blood vessels and depends on a functional, quiescent endothelial monolayer. However, how endothelial cells enter and maintain quiescence in the presence of angiogenic factors is still poorly understood. Here we identify the fibroblast growth factor (FGF) antagonist Sprouty2 (Spry2) as a key player in mediating endothelial quiescence and barrier integrity in mouse aortic endothelial cells (MAECs): Spry2 knockout MAECs show spindle-like shapes and are incapable of forming a functional, impermeable endothelial monolayer in the presence of FGF2. Whereas dense wild type cells exhibit contact inhibition and stop to proliferate, Spry2 knockout MAECs remain responsive to FGF2 and continue to proliferate even at high cell densities. Importantly, the anti-proliferative effect of Spry2 is absent in sparsely plated cells. This cell density-dependent Spry2 function correlates with highly increased Spry2 expression in confluent wild type MAECs. Spry2 protein expression is barely detectable in single cells but steadily increases in cells growing to high cell densities, with hypoxia being one contributing factor. At confluence, Spry2 expression correlates with intact cell-cell contacts, whereas disruption of cell-cell contacts by EGTA, TNFα and thrombin decreases Spry2 protein expression. In confluent cells, high Spry2 levels correlate with decreased extracellular signal-regulated kinase 1/2 (Erk1/2) phosphorylation. In contrast, dense Spry2 knockout MAECs exhibit enhanced signaling by Erk1/2. Moreover, inhibiting Erk1/2 activity in Spry2 knockout cells restores wild type cobblestone monolayer morphology. This study thus reveals a novel Spry2 function, which mediates endothelial contact inhibition and barrier integrit

A bioluminescent mouse model of pancreatic β-cell carcinogenesis

The Rip1Tag2 transgenic mouse model of pancreatic β-cell carcinogenesis has been instrumental in identifying several hallmarks of cancer, including tumor cell evasion from apoptosis, tumor angiogenesis and tumor invasion. Moreover, Rip1Tag2 mice have been helpful in the development and testing of innovative cancer therapies and tumor imaging protocols. However, based on tumor localization in the mouse, primary tumor growth and metastatic dissemination cannot be easily monitored in a longitudinal axis by non-invasive and low-technology approaches. Here, we report the generation of a new transgenic mouse line as a versatile tool to study β-cell carcinogenesis. Transgenic expression of a bicistronic messenger RNA encoding simian virus large T antigen and firefly luciferase in pancreatic β-cells recapitulates insulinoma development in a reproducible multistage process. In the mouse line called RipTag-IRES-Luciferase line (RTL) 1, the β-cell-specific expression of luciferase allows the non-invasive monitoring of primary tumor growth over time in vivo and the detection and quantification of disseminated tumor cells and micrometastases in distant organs ex vivo. When crossed to mouse lines in which the expression of cancer ‘modifier' genes has been manipulated, tumor initiation and tumor progression are similarly affected as previously reported for Rip1Tag2 mice, indicating a robust tumor progression pathway shared between the two different transgenic mouse lines. Together, the data indicate that the RTL1 mouse line will be of great value to study anti-tumoral therapeutic approaches as well as to define the functional roles of cancer- and metastasis-related genes when crossed to appropriate transgenic or gene-targeted mouse line

An immature B cell population from peripheral blood serves as surrogate marker for monitoring tumor angiogenesis and anti-angiogenic therapy in mouse models

Tumor growth depends on the formation of new blood vessels (tumor angiogenesis) either from preexisting vessels or by the recruitment of bone marrow-derived cells. Despite encouraging results obtained with preclinical cancer models, the therapeutic targeting of tumor angiogenesis has thus far failed to deliver an enduring clinical response in cancer patients. One major obstacle for improving anti-angiogenic therapy is the lack of validated biomarkers, which allow patient stratification for suitable treatment and a rapid assessment of therapy response. Toward these goals, we have employed several mouse models of tumor angiogenesis to identify cell populations circulating in their blood that correlated with the extent of tumor angiogenesis and therapy response. Flow cytometry analyses of different combinations of cell surface markers that define subsets of bone marrow-derived cells were performed on peripheral blood mononuclear cells from tumor-bearing and healthy mice. We identified one cell population, CD45dimVEGFR1⁻CD31low, that was increased in levels during active tumor angiogenesis in a variety of transgenic and syngeneic transplantation mouse models of cancer. Treatment with various anti-angiogenic drugs did not affect CD45dimVEGFR1⁻CD31low cells in healthy mice, whereas in tumor-bearing mice, a consistent reduction in their levels was observed. Gene expression profiling of CD45dimVEGFR1⁻CD31low cells characterized these cells as an immature B cell population. These immature B cells were then directly validated as surrogate marker for tumor angiogenesis and of pharmacologic responses to anti-angiogenic therapies in various mouse models of cancer

Temporal effects of Sprouty on lung morphogenesis

AbstractParacrine signaling mediated by FGF-10 and the FGF-R2IIIb receptor is required for formation of the lung. To determine the temporal requirements for FGF signaling during pulmonary morphogenesis, Sprouty-4 (Spry-4), an intracellular FGF receptor antagonist, was expressed in epithelial cells of the fetal lung under control of a doxycycline-inducible system. Severe defects in lobulation and severe lung hypoplasia were observed when Spry-4 was expressed throughout fetal lung development (E6.5–E18.5) or from E6.5 until E13.5. Effects of Spry-4 on branching were substantially reversed by removal of doxycycline from the dam at E12.5, but not at E13.5. In contrast, when initiated late in development (E12.5 to birth), Spry-4 caused less severe pulmonary hypoplasia. Expression of Spry-4 from E16.5 to E18.5 reduced lung growth and resulted in perinatal death due to respiratory failure. Expression of Spry-4 during the saccular and alveolar stages, from E18.5 to postnatal day 21, caused mild emphysema. These findings demonstrate that the embryonic-pseudoglandular stage is a critical time period during which Spry-sensitive pathways are required for branching morphogenesis, lobulation, and formation of the peripheral lung parenchyma

Tracking and characterization of partial and full epithelial-mesenchymal transition cells in a mouse model of metastatic breast cancer

The various stages of epithelial-mesenchymal transition (EMT) generate phenotypically heterogeneous populations of cells. Here, we detail a dual recombinase lineage tracing system using a transgenic mouse model of metastatic breast cancer to trace and characterize breast cancer cells at different EMT stages. We describe analytical steps to label cancer cells at an early partial or a late full EMT state, followed by tracking their behavior in tumor slice cultures. We then characterize their transcriptome by five-cell RNA sequencing. For complete details on the use and execution of this protocol, please refer to Luond et al. (2021)