Article Text
PDF
Statistics from Altmetric.com
Introduction
Early recurrence is a frequent cause of poor survival after hepatocellular carcinoma (HCC) resection. At the cell level, efforts to link HCC with the dysfunction of signalling pathways identified several alterations, especially in the Wnt/β-catenin pathway.1 Wnt signalling controls the balance between cell proliferation and differentiation and its regulation relies partly on subcellular compartmentalisation at the plasma membrane in late endosomes and/or on the cytoskeleton (see refs. 2 ,3 for reviews) (figure 1A).
The Wnt/β-catenin pathway: regulation by subcellular compartmentalisation and links with the cell cycle through the involvement of Protein Regulator of Cytokinesis 1 (PRC1). (A) (1) The β-catenin destruction complex, which comprises adenomatous polyposis coli (APC), Axin, glycogen synthase kinase 3β (GSK3β) and casein kinase 1 (CK1), functions to phosphorylate free β-catenin, allowing its recognition by the E3 enzyme β-Transducin repeats-Containing Protein (β-TrCP) and its consecutive degradation by the proteasome. (2) The lipoprotein receptor-related protein (LRP)5/6 signalosome: Wnt agonist binding to its receptor complex (Frizzled+LRP5/6) allows LRP5/6 phosphorylation, Axin and Microtubule-Actin Crosslinking Factor 1 (MACF1) recruitment to the signalosome and then inactivation of GSK3β. (3) APC motorisation towards the microtubule plus ends allows its interaction with end-binding 1 (EB1) and mDia to stabilise microtubules at the migrating cell front. (4) Cytoplasmic PRC1 binds to microtubule bundles and interacts with the destruction complex to inactivate it. (5) Inactivation of the destruction complex leads to β-catenin nuclear entry and association with the T-cell factor/lymphoid enhancer factor (TCF/LEF) transcription factor to promote gene expression. (6) Nuclear localisation of Axin, APC, Dishevelled (Dvl) and PRC1 modulates the Wnt/β-catenin signalling pathway. Nuclear APC competes with TCF/LEF for binding to β-catenin and favours its nuclear export. Nuclear Axin binds to the promoter region and competes with the binding of the β-catenin transcription complex on the promoter of target genes like MYC. (7) Wnt/β-catenin-dependent stimulation of gene expression in hepatocellular carcinoma (HCC) leads to PRC1 and several other Wnt-regulated recurrence-associated genes (WRRAGs) overexpression. (B) List and functions of WRRAGs-coded proteins and PRC1-associated proteins. The WRRAGs that correlate with a high level of PRC1 expression are indicated in red. Proteins directly interacting with PRC1 are indicated on a grey background. Those that are not WRRAGs are underlined. CPC stands for chromosome passenger complex, and HCCr stands for HCC recurrence. CDK, cyclin-dependent kinase; MT, microtubule; NDC, nuclear division cycle; RZZ, rod-zw10-zwilch; SMC, structural maintenance of chromosomes.
In this issue of Gut, Chen and collaborators4 identified the microtubule-associated Protein Regulator of Cytokinesis 1 (PRC1/Microtubule-Associated Protein 65 (MAP65)/anaphase spindle elongation 1 (Ase 1)) as a new factor that is overexpressed in HCC and that promotes recurrence. Deciphering the role PRC1 plays in cell proliferation and migration led to determine how PRC1 activates the Wnt/β-catenin pathway and how its expression is controlled by Wnt signalling. Furthermore, this study correlates PRC1 overexpression in recurrent HCC with Wnt-mediated expression of specific genes (WRRAGs, Wnt-regulated recurrence-associated genes) (figure 1B). Here, we present a brief overview of the actors of the Wnt/β-catenin pathway and of PRC1, their relation with the microtubule cytoskeleton and the way they could contribute to HCC recurrence mechanisms.
The WNT/β-catenin pathway
Canonical Wnt signalling
This pathway2 ,5 (figure 1A) relies on the stabilisation and on the localisation of β-catenin, which is both a component of cadherin-based junctions and a coactivator of the T-cell factor/lymphoid enhancer factor (TCF/LEF) transcription factors. The degradation of excess β-catenin depends on the activity of a destruction complex. This complex mainly comprises the two antioncogenes Axin and adenomatous polyposis coli (APC), the two kinases glycogen synthase kinase 3β (GSK3β) and casein kinase 1 (CK1), which both phosphorylate β-catenin to allow its ubiquitylation by the E3-ubiquitin ligase β-Transducin repeats-Containg Protein (β-TrCP) and its consecutive degradation by the proteasome.
Wnt agonists binding to the Frizzled receptor and to its coreceptor (the low density lipoprotein receptor-related protein LRP5/6) activate LRP5/6 phosphorylation by GSK3β and CK1. This modification triggers the recruitment of the scaffolding protein Axin to the so-called LRP5/6 signalosome via the adaptor Dishevelled (Dvl) and the Microtubule-Actin Crosslinking Factor 1/Actin Crosslinking Family 7 (MACF1/ACF7),6 and hence inhibits GSK3β. Further trafficking of the signalosomes to the late endocytic pathway contributes to GSK3β sequestration and inhibition, leading to β-catenin stabilisation and nuclear translocation. There, β-catenin upregulates the expression of several genes, many of which allow progression through the cell cycle.
Links between the Wnt/β-catenin pathway and the microtubule cytoskeleton
Actors of the Wnt pathway have been proposed to regulate the dynamics of microtubules.3 APC, with its partners end-binding 1 (EB1) and the formin mDia, can stabilise microtubules oriented towards the front end of migrating cells.7 APC can also target MACF1 to the cell cortex to capture microtubule ends.8 Additionally, GSK3β and CK1 can phosphorylate several microtubule-associated proteins (tau, MAP1B, MAP2), leading to microtubule destabilisation (reviewed in ref. 3), while, upon GSK3β inhibition mediated by Dvl, Axin can bind to and stabilise microtubules.9
Reciprocally, microtubules may affect Wnt signalling by the spatiotemporal regulation of the destruction complex. Indeed, Dvl, Axin, APC and GSK3β can associate with microtubules,3 while the active destruction complex (in which APC is phosphorylated by GSK3β) cannot.10 Chen et al 4 now reveal that upon Wnt stimulation, APC partially translocates to microtubules together with PRC1, Axin and GSK3β. These interactions with the cytoskeleton may sequester the destruction complex, thus contributing to inhibit β-catenin degradation.
The microtubule-associated protein PRC1/MAP65/Ase 1 and HCC
PRC1/MAP65/Ase 1 (not to be confused with the polycomb repressor complex 1) is a conserved nuclear protein required during anaphase for central-spindle assembly and for completion of cytokinesis. PRC1 homodimers bind to antiparallel microtubule overlaps in the central part of the spindle, bundle microtubules and then recruit and concentrate factors to the spindle midzone/midbody during anaphase and cytokinesis.11 ,12
Chen et al 4 revealed that PRC1 is overexpressed in HCC and early recurrent HCC and accumulates ectopically in the cytosol of interphase cells. Upon Wnt activation in interphase cells, PRC1 exits the nucleus and relocalises to the plasma membrane and to microtubules. There, PRC1 binding to the Wnt degradation complex triggers both the sequestration of the complex and the decrease of cellular APC. Ultimately, PRC1 contributes to stimulate the expression of a subset of WRRAGs, out of which 43 correlate with high PRC1 expression. Strikingly, at least 24 of these 43 genes code proteins involved in mitosis together with PRC1 (figure 1B).
In parallel to β-catenin stabilisation, GSK3β inhibition is known to cause the STabilization Of Proteins (Wnt/STOP pathway) that mediate the increase in cell size before mitosis.13 It is likely that PRC1 overexpression in HCC recurrence could also promote such stabilisation of proteins. Together, Wnt/STOP and WRRAG-coded proteins build-up would provide HCC cells with the molecular equipment required to proliferate.
Nuclear compartmentalisation and sequestration of the proteins involved in controlling the WNT/β-catenin pathway
APC and Axin compartmentalisation is an important regulator of the Wnt pathway function (figure 1A). Nuclear APC localisation (reviewed in ref. 14) is necessary for its tumour suppression effect. APC exit from the nucleus can promote the nuclear export of β-catenin, while nuclear APC can attenuate Wnt-mediated transcription by sequestering β-catenin. Similarly, nuclear Axin dampens nuclear β-catenin transcriptional effects.15 In contrast, when Dvl is in the nucleus, it can mediate the formation of a Dvl–c-Jun–β-catenin–TCF complex, allowing the activation of Wnt target genes transcription.16
In interphase mammalian cells, PRC1 is mostly nuclear.4 ,17 Chen et al 4 show that ectopic cytoplasmic targeting of PRC1 causes a high level of microtubule bundling and stimulates Wnt signalling. Therefore, the observed PRC1 mislocalisation in HCC cells could be sufficient to bundle microtubules and to recruit the degradation complex to stabilise β-catenin.
Concluding remarks and future prospects
Constitutive activation of the Wnt pathway is frequently found in hepatocellular adenomas and in HCC (see refs. 18 ,19 for reviews). Chen et al reveal that the upregulation of PRC1, a new Wnt pathway activator, also correlates with HCC recurrence. As it is both a regulator of the destruction complex and a target of β-catenin/LEF-mediated gene regulation, PRC1 functions in an amplification loop at the crossroads of directional cell migration/invasion and of cell proliferation. As such, it might be a valuable cellular marker of HCC that could be predictive of recurrence, but more strategically, it could be viewed as a potential therapeutic target.
Several inhibition strategies of the Wnt pathway have already been investigated either to perturb the interaction between Frizzled and Wnt, to stimulate the activity of the destruction complex or to inhibit β-catenin-mediated control of gene expression.20 A new interesting strategy would consist in preventing the homodimerisation of PRC1. This would prevent the recruitment and the inhibition of the destruction complex to microtubule bundles in interphase cells and at the same time, it would function to alter the build-up of the central-spindle complex and hopefully prevent mitosis completion in proliferating cells. Targeting such an inhibitor via a vectorisation system that could be cleared from the bloodstream by the liver could efficiently play the role of a two-stage rocket against HCC cells.
References
Footnotes
Contributors Both authors contributed to article design and writing.
Competing interests None declared.
Provenance and peer review Commissioned; internally peer reviewed.