Cadherin Switch during EMT in Neural Crest Cells Leads to Contact Inhibition of Locomotion via Repolarization of Forces

Contact inhibition of locomotion (CIL) is the process through which cells move away from each other after cell-cell contact, and it contributes to malignant invasion and developmental migration. Various cell types exhibit CIL, whereas others remain in contact after collision and may form stable junctions. To investigate what determines this differential behavior, we study neural crest cells, a migratory stem cell population whose invasiveness has been likened to cancer metastasis. By comparing pre-migratory and migratory neural crest cells, we show that the switch from E- to N-cadherin during EMT is essential for acquisition of CIL behavior. Loss of E-cadherin leads to repolarization of protrusions, via p120 and Rac1, resulting in a redistribution of forces from intercellular tension to cell-matrix adhesions, which break down the cadherin junction. These data provide insight into the balance of physical forces that contributes to CIL in cells in vivo.</p

Paracrine regulation of neural crest EMT by placodal MMP28.

Epithelial-mesenchymal transition (EMT) is an early event in cell dissemination from epithelial tissues. EMT endows cells with migratory, and sometimes invasive, capabilities and is thus a key process in embryo morphogenesis and cancer progression. So far, matrix metalloproteinases (MMPs) have not been considered as key players in EMT but rather studied for their role in matrix remodelling in later events such as cell migration per se. Here, we used Xenopus neural crest cells to assess the role of MMP28 in EMT and migration in vivo. We show that a catalytically active MMP28, expressed by neighbouring placodal cells, is required for neural crest EMT and cell migration. We provide strong evidence indicating that MMP28 is imported in the nucleus of neural crest cells where it is required for normal Twist expression. Our data demonstrate that MMP28 can act as an upstream regulator of EMT in vivo raising the possibility that other MMPs might have similar early roles in various EMT-related contexts such as cancer, fibrosis, and wound healing

Development of a Conditional Bioluminescent Transplant Model for TPM3-ALK-Induced Tumorigenesis as a Tool to Validate ALK-Dependent Cancer Targeted Therapy.

Overexpression and activation of TPM3-ALK tyrosine kinase fusion protein is a causal oncogenic event in the development of Anaplastic Large Cell Lymphoma and Inflammatory Myofibroblastic ALK-positive tumours. Thus, the development of ALK specific tyrosine kinase inhibitors is a current therapeutic challenge. Animal models are essential to assess, in vivo, the efficiency of ALK-oncogene inhibitors and to identify new and/or additional therapeutic targets in the ALK tumorigenesis pathway. Using the tetracycline system to allow conditional and concomitant TPM3-ALK and luciferase expression, we have developed a unique transplant model for bioluminescent TPM3-ALK-induced fibroblastic tumours in athymic nude mice. The reversible TPM3-ALK expression allowed us to demonstrate that this oncogene is essential for the tumour growth and its maintenance. In addition, we showed that this model could be used to precisely assess tumour growth inhibition upon ALK chemical inactivation. As proof of principle, we used the general tyrosine kinase inhibitor herbimycin A to inhibit ALK oncoprotein activity. As expected, herbimycin A treatment reduced tumour growth as assessed both by tumour volume measurement and bioluminescent imaging. We conclude that this transplant model for TPM3-ALK-induced tumours represents a valuable tool not only to accurately and rapidly evaluate in vivo ALK-targeted therapies but also to gain insight into the mechanism of ALK-positive tumour development

Rescue of Sox10 expression can occur in absence of a rescue of Six1 expression.

(a–f) In situ hybridization for Sox10 (a–c) and Six1 (d–f), in embryos injected with CMO (a, n = 29; d, n = 56), MMP28-MOspl (b, n = 40; e, n = 43), or co-injected with MMP28-MOspl and mRNA for MMP28wt-GFP (c, n = 31; f, n = 41). (g, h) Proportions of embryos with symmetrical or decreased expression of Sox10 (g) or Six1 (h) in each experimental condition. Contingency tables for comparison of proportions: Sox10 CMO vs. MOspl, T = 57.34 (***), MOspl vs. rescue condition, T = 36.65 (***); Six1 CMO vs. MOspl, T = 70.59 (***), MOspl vs. rescue condition, T = 7.7 (***), Rescue Sox10 vs. Rescue Six1, T = 19.5 (***). Asterisks on images indicate the injected side. Dotted lines mark the midline of each embryo. Brown arrows indicate the missing portion of Six1 expression domain in MMP28-MOspl and MMP28-MOspl+mRNA MMP28wt-GFP embryos. Scale, embryos are 500 μm wide on average. (TIF)</p

3D confocal imaging of neural crest cells expressing MMP28-GFP.

Neural crest cells express MMP28-GFP (green) and are counterstained with DAPI (blue). DAPI staining is used as a mask to sample the green channel, highlighting the amount of MMP28-GFP present in the nuclei. Related to Fig 4. (AVI)</p

Twist expression is sufficient to rescue adhesion and migration of neural crest cells after MMP28 knockdown.

(a) Diagram depicting the experimental set-up with injection of MO and mRNA in 2 blastomeres on one side of 8-cell stage embryos and the embryos analysed at neural crest migration stage (stage 23). (b) In situ hybridization against foxd3 following injection with CMO, MMP28-MOspl, or the co-injection of MMP28-MOspl together with twist or cadherin-11 mRNA. (c) Graph plotting the distance migrated by neural crest cells in the experimental conditions shown in (b), nCMO = 25, nMMP28-MOspl = 31 nMMP28-MOspl+twist = 57, nMMP28-MOspl+cad11 = 47 from 2 independent experiments. ANOVA followed by uncorrected Fisher’s LSD; ****, p p = 0.8829. (d) Diagram depicting the experimental procedure for neural crest culture on Fibronectin. (e) Low magnification images of explants in all experimental conditions after fixation. Adhering explants are outlined in purple, detached explants are outlined in yellow; scale bar, 500 μm. (f) Quantification of adhering explants, CMO = 13/13; MMP28-MOspl = 5/13; MMP28-MOspl+twist = 12/12; MO28-MOspl+cad11 = 8/10. Contingency tables for the comparison of proportion; CMO vs. MMP28-MOspl, T = 10.53, alpha 0.01 (**), MMP28-MOspl vs. MMP28-MOspl+twist, T = 9.88, alpha 0.01 (**), MMP28-MOspl vs. MMP28-MOspl+cad11, T = 3.31 (ns), CMO vs. MMP28-MOspl+cad11, T = 2.85, (ns). (g) DAPI (magenta) and Phalloidin (white) staining; scale bars, 40 μm for clusters, 20 μm for single cells. (h) Protrusion area in μm2, outward protrusions CMO (n = 64), MMP28-MOspl (n = 49), MOspl+twist (n = 47), MOspl+cad11 (n = 87); cryptic protrusions CMO (n = 103), MMP28-MOspl (n = 29), MOspl+twist (n = 140), MOspl+Cad11 (n = 100); ANOVA, Kruskal–Wallis test; ****, p n = 12), MMP28-MOspl (n = 10), MOspl+twist (n = 12), MOspl+cad11 (n = 10). (l) Speed of individual cells (μm/min) from each experimental conditions shown in panels (i–k); CMO (n = 30), MMP28-MOspl (n = 30), MOspl+twist (n = 32), MOspl+cad11 (n = 28). ANOVA followed by multiple comparisons; CMO vs. MOspl p = 0.0057 (**), CMO vs. MOspl+cadherin11 p = 0.0291 (*), CMO vs. MOspl+twist p = 0.2483 (ns). Numerical data from all graphs can be found in the supporting S1 Data file. CMO, control Morpholino; MMP, matrix metalloproteinase.</p