Basal extrusion drives cell invasion and mechanical stripping of E-cadherin

Background

Oncogenesis can start with a mutation to a single cell, resulting in a transformed malignant cell, within a healthy epithelial sheet. It is increasingly apparent that single transformed cells can be detected and squeezed out of the tissue by the surrounding normal neighbours. This process has been observed in flies, cell lines, organoids, zebrafish and in vivo using mouse models (Bielmier et al., 2016; Hogan et al., 2009; Kon et al., 2017; Anton et al., 2018).

However, what is less clear is the fate of extruded cells. It is currently thought that the direction of extrusion can impact an extruded cells’ fate; if a cell is pushed out apically then it dies or is cleared through the lumen. However, it is unclear what happens to cells that extrude basally.

In this preprint, the authors investigate the fate of KRas-transformed cells surrounded by normal neighbours in zebrafish. Expression of different oncogenes was combined with fluorescent markers to track mutant cells in real-time. Oncogenes which promote extrusion are associated with metastatic cancers so the authors hypothesise that basal extrusion could be a novel mechanism of metastasis.

Key findings of the preprint

KRas-transformed cells can be extruded basally and apically and initiate tumourigenesis.

Firstly, the authors injected EGFP-KRasV12 plasmids driven by the krt4 promoter into one-cell embryos to generate mosaic expression of KRas in epidermal cells. They found that this was sufficient to form cell masses in 58% of zebrafish embryos compared to 1% of EGFP-CAAX controls. Time-lapse imaging of labelled cells over time revealed that KRasV12 cells can extrude both apically and basally, although significantly more basal extrusions were observed. They saw that basal extrusion can lead to invasion and internalisation of mutant cells into different parts of the zebrafish. Although KRas-transformed cells could form cell masses, a large number of mutant cells were lost over time, which could be a result of extrusion observed in time-lapse imaging.

Loss of p53 promotes the survival extruded cells.

During the evolution of cancer, transformed cells accumulate additional mutations which drive tumourigenesis. To model this the authors combined expression of KRasV12 with depletion of p53 via a translation-blocking morpholino. This led to a reduction in basal extrusion of aberrant cells. However, loss of p53 increased the survival of basally invading cells and their ability to survive as internalised masses at later time points.

Extruded cells can spread through the bloodstream and may initiate EMT

After establishing the system, the authors began characterising cells that invade basally. They found that all internalised cells lose the epithelial marker E-cadherin and a small subset expressed the mesenchymal marker N-cadherin. Intriguingly, time lapse imaging in combination with a vasculature reporter was used to identify GFP-KRasV12 cells in the bloodstream with 65% of 42 zebrafish having circulating cells. Finally, using different fluorescent markers for the apical and cytoplasm it was observed that the apical membrane is ripped off during extrusion and the remainder of the cell migrates away. The authors suggest that basal extrusion drives invasion of KRasV12 cells and strips off the apical surfaces and associated E-cadherin.

What I like about this preprint

This preprint demonstrates that extrusion can lead to mutant cells in the circulation of zebrafish, which is a possible new way for oncogenic cells to spread around the body. Although lots of work on the mechanism of extrusion has been carried out, this paper begins to characterise the fate of extruded cells.

The authors looked at how the mechanical force of KrasV12 cells being squeezed out constricts the nucleus and can scrape off the E-cadherin apical membrane proteins. This sudden loss of the apical membrane could have important implications for metastasis and EMT.

I also liked the great videos of cells extruding from zebrafish!

Questions to the authors

What do you think could be regulating whether mutant cells either accumulate into masses or extrude in the different parts of the zebrafish?

In the movies do you ever see KRasV12 cells extravasate from the circulation?

Mechanical stretch can promote YAP translocation into the nucleus, have you looked for YAP nuclear localisation in extruding cells?

References:

Anton, K. A., Kajita, M., Narumi, R., Fujita, Y., & Tada, M. (2018). Src-transformed cells hijack mitosis to extrude from the epithelium. Nature communications, 9(1), 4695.

Bielmeier, C., Alt, S., Weichselberger, V., La Fortezza, M., Harz, H., Jülicher, F., … & Classen, A. K. (2016). Interface contractility between differently fated cells drives cell elimination and cyst formation. Current Biology, 26(5), 563-574.

Hogan, C., Dupré-Crochet, S., Norman, M., Kajita, M., Zimmermann, C., Pelling, A. E., … & Hosoya, H. (2009). Characterization of the interface between normal and transformed epithelial cells. Nature cell biology, 11(4), 460.

Kon, S., Ishibashi, K., Katoh, H., Kitamoto, S., Shirai, T., Tanaka, S., … & Kamasaki, T. (2017). Cell competition with normal epithelial cells promotes apical extrusion of transformed cells through metabolic changes. Nature cell biology, 19(5), 530.

Tags: cell competition, extrusion, zebrafish

Posted on: 2nd January 2019

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