Control of spindle orientation by Axin in 3D intestine and liver organoid cultures


Supervising PI

Trevor Dale (website)

ESR12 Eider Valle Encinas

Project Description

We aim to determine whether the cancer-inducing loss of Axin1 and Axin2 proteins in the liver is mediated by changes to cell polarity.

3D liver organoid cultures generated from mice with Cre-loxP flanked alleles of Axin1 and Axin2 will be used to study the effects of deleting both anti-oncogenes. Cell biological, biochemical and gene expression studies will be used to identify the mechanisms by which Axin loss leads to cellular transformation. A particular focus will be paid to alterations in mitotic spindle orientation since Axin and its binding partners APC, b-catenin and GSK3 have previously been shown to associate with components of the centrosomal complexes and deregulation of physiological spindle-orientation in stem cell vs. progenitor decisions has been suggested to be a key step in oncogenesis.

Objectives

To determine whether the cancer-inducing loss of Axin1 and Axin2 proteins in the liver is mediated by changes to cell polarity.

Summary of Results

Eider Valle Encinas carried out a detailed series of molecular and histological analyses of biliary-derived organoids to investigate their utility as an in vitromodel of hepatocyte biology. She demonstrated that the organoids were unable to recapitulate any aspect of metabolic zonation; a key feature that is regulated by Wnt signalling in the liver lobule. This work will be an important corrective for the organoid field since it definitively demonstrates that the organoids should not be considered to be ‘liver organoids’. By contrast and in collaboration with the Beatson Institute and Edinburgh University, the ESR identified many similarities between the organoid system and the differentiative processes that rescue liver function following chronic liver damage. The ESR also used primary hepatocytes and a novel organoid system in which true hepatocytes are maintained in culture to demonstrate Wnt-dependent regulation of zonation markers including selected cytochrome markers and Axin2. In this work, the ESR showed that the regulation of hepatocyte function could be controlled by Wnt- and R-spondin- conjugated beads, recapitulating in vivo interactions between endothelial cells of the Central Vein and hepatocytes.

References

  1. Feng GJ, Cotta W, Wei XQ, Poetz O, Evans R, Jardé T, et al. Conditional disruption of Axin1 leads to development of liver tumors in mice. Gastroenterology. 2012 Dec;143(6):1650–9.
  2. Jardé T, Evans RJ, McQuillan KL, Parry L, Feng GJ, Alvares B, et al. In vivo and in vitro models for the therapeutic targeting of Wnt signaling using a Tet-OΔN89β-catenin system. Oncogene. 2013 Feb 14;32(7):883–93.
  3. Dale T, Clarke PA, Esdar C, Waalboer D, Adeniji-Popoola O, Ortiz-Ruiz M-J, et al. A selective chemical probe for exploring the role of CDK8 and CDK19 in human disease. Nat Chem Biol. 2015 Oct 26;