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Mechanisms of tumorigenesis and tumor progression Antonio García de Herreros

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The role of the deubiquitinases (DUBs) involved in regulating Snail stability in the chemo-resistance of tumour cells

Directed by Dr. Víctor M. Díaz

Our group is interested in looking at the EMT (Epithelial to mesenchymal transition), a process that occurs during both embryonic development and in tumour cells. The EMT is characterised by the loss of epithelial markers, such as E-cadherin, and the gain of mesenchymal markers, such as fibronectin. The Snail1 transcription factor is essential for triggering the EMT during tumour progression. Snail1 is a highly unstable protein, due to its rapid ubiquitination and degradation by proteasome. Ubiquitination requires the successive action of the enzyme E1, or ubiquitin activator; E2, or ubiquitin conjugator; and E3 enzymes, or ubiquitin ligases. Our group has characterised the F-box protein known as FBXL14 as an essential part of an E3 ubiquitination complex (reviewed in Díaz VM et al., 2014). Thanks to a "screening" utilising a shRNA library we have also characterised FBXL5 as the E3 ubiquitin ligase that interacts with Snail1 in the nucleus. Snail1 and FBXL5 bind through the C-terminal region of Snail1, favouring their degradation. While FBXL14 is cytoplasmic and ubiquitinises Snail1 in lysines K98, K137, and K146, FBXL5 is found in the nucleus and is capable of poly-ubiquitinising Snail1 in lysines K85, K146 and K234 (Figure 1).

A new, unexpected function of FBXL5 is its ability to modulate the levels of Snail1 in chromatin, as the ubiquitination of K234 in Snail1 decreases its binding with DNA. With regard to regulation, FBXL14 levels decrease during the intratumoural hypoxia of colon adenocarcinomas, causing Snail1 to stabilise and inducing EMT. In contrast, FBXL5 levels are affected by gamma radiation, which results in Snail1 stabilisation under these conditions (Viñas-Castells et al, 2014).

Figure 1: Diagrams of the Snail1-Fbxl5 interaction.

The stability of Snail1 is regulated by GSK3β phosphorylation, which induces its degradation (reviewed in Díaz et al., 2016). Endothelial cells activated by Notch suffer an endothelial-mesenchymal transition (or EndMT). EndMT is a special case of EMT in which the Akt2 kinase plays an important role, as it phosphorylates GSK3β deactivating it, causing an increase in Snail1 levels. Interestingly, during EndMT there is a "switch" between the two isoforms of Akt, inhibiting Akt1 and stimulating Akt2, which in addition is translocated to the nuclear membrane where it is activated (Figure 2) (Frías A, et al., 2015).

  

Figure 2. Notch activation promotes EndMT thanks to the location of AKT2 in the nuclear membrane.

Our most recent research focuses on the deubiquitination of Snail1 by deubiquitinating enzymes (or DUBs). We "screened" siRNA libraries to find the Snail1 DUBs and determine their involvement in tumoural proliferation, migration and invasion (Lambies et al, in preparation). In addition, we are studying the relationship between resistance to platinum treatment developed by tumour cells, the activation of DUBs, and the stabilisation of Snail1.

The four most recent publications on this topic are:

  • Díaz VM*, Viñas-Castells R, García de Herreros A*. Regulation of the protein stability of EMT transcription factors. Cell Adh Migr. 2014. 8: 418-428.
  • Viñas-Castells R, Frías Á, Robles-Lanuza E, Zhang K, Longmore GD, García de Herreros A*, Díaz VM*. Nuclear ubiquitination by FBXL5 modulates Snail1 DNA binding and stability. Nucleic Acids Res. 2014. 42: 1079-1094.
  • Frías A, Lambies G, Viñas-Castells R, Martínez-Guillamon C, Dave N, García de Herreros A*, Díaz VM*. A Switch in Akt Isoforms Is Required for Notch-Induced Snail1 Expression and Protection from Cell Death. Mol Cell Biol. 2015. 36: 923-940.
  • Díaz VM*, García de Herreros A*. F-box proteins: Keeping the epithelial-to-mesenchymal transition (EMT) in check. Semin Cancer Biol. 2016. 36: 71-79.

(*Corresponding authors)

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