Focusing on beta‐catenin activating mutations to refine liver tumor profiling
Authors: Sabine Colnot
Journal: Hepatology (2016)

Potential conflict of interest: Nothing to report. See Article on page 2047 The last two decades have seen the emergence of β‐catenin as a fundamental and dual signaling molecule in the adult liver: Through its limited activation to the pericentral area, it patterns the so‐called hepatic metabolic zonation, and Wnt/R‐Spondin‐activated pericentral hepatocytes are proposed to be essential for liver homeostasis.1 When activated by oncogenic mutations, β‐catenin aberrant signaling leads to liver tumorigenesis.2 These mutations are mainly targeted to the CTNNB1 gene encoding β‐catenin and affect three liver primary tumors: hepatocarcinomas (HCCs), hepatocellular adenomas (HCAs), and hepatoblastomas (HBs). Among these, the main health issue concerns HCCs, the second‐leading cause of cancer death worldwide, one third of which occur with CTNNB1‐activating mutations.3CTNNB1‐mutated HCCs appear either in a healthy liver or they are correlated to a context of alcoholism and are found mostly in the absence of an underlying hepatitis B virus infection. In this issue of Hepatology, Rebouissou, Franconi, et al. revisit CTNNB1‐mutated HCCs, also showing their increased occurrence in a setting of metabolic syndrome.3 More interestingly, they also found that the spectrum of CTNNB1 mutations depends on the benign (HCA) or malignant (HCC) status of the liver tumor. They finally highlight the need for a "bedside to bench" turnaround as they touch on some weakness regarding basic knowledge of how Wnt/β‐catenin signaling works. The physiological β‐catenin signaling cascade is directed by Wnts, together with their coactivators, R‐Spondins (Fig. 1A). In their absence, cytosolic β‐catenin is carried out by a so‐called "degradation complex" constituted from the tumor suppressors, adenomatous Polyposis Coli (APC) and AXIN, and from kinases. Within this complex, the paradigm is that β‐catenin gets a priming phosphorylation on Serine‐45 by the CK1 kinase (Fig. 1B). This allows its subsequent phosphorylations on adjacent serine/threonine residues (S33, S37, and T41) by the glycogen synthase kinase 3 (GSK3) kinase. The beta‐transducin repeat containing E3 ubiquitin protein ligase (β‐TrCP) thereafter recognizes the DSGIHS box when phosphorylated (residues 32‐37) as a prerequisite for β‐catenin ubiquitination and proteasomal degradation. The Wnt/R‐Spondin ligands activate β‐catenin signaling: They bind their receptors, FRIZZLED and leucine‐rich repeat containing G‐protein‐coupled receptor 4/5 (LGR4/5), leading to AXINs recruitment. This disrupts the degradation complex and impairs β‐catenin phosphorylation, leading to its stabilization and nuclear translocation, making it become a cofactor with the lymphoid enhancer factor/T‐cell factor (LEF/TCF) family of transcription factors and controlling downstream targets responsible for oncogenicity. Classically, activating mutations of β‐catenin signaling either inactivate APC or AXIN tumor‐suppressor genes or stabilize β‐catenin by rendering it unfit for phosphorylation/degradation, through mutations of the residues located in its exon 3. These can be missense mutations leading to a lack of phosphorylation of the DSGIHS box or intragenic deletions encompassing the exon 3, all of these allowing the translation of a constitutively stable β‐catenin. Whereas APC mutation is the main mutational event in colorectal cancer, mutations of CTNNB1 and AXIN1, at a lesser extent, are the main ones in liver cancers.Figure 1: Physiological and pathological β‐catenin signalings. (A) The Wnt/Spondin pathway is physiologically activated only in pericentral hepatocytes; see a previous work1 for more information. (B) Localization of CTNNB1 mutations along β‐catenin protein. (C) Occurrence of distinct CTNNB1 mutations in HCAs and HCCs.This multiplicity of mutational events affecting β‐catenin signaling was a first level of complexity in liver cancers. The cause of the low frequency of APC mutations in liver cancers is still unknown, even if, in mice, modeling these mutations are fully efficient to generate HCCs with strong similarities with CTNNB1‐mutated human HCCs.4 On the other hand, AXIN1‐mutated HCCs are rather frequent, but are classified distinctly from CTNNB1‐mutated ones, with distinct transcriptomic characteristics, suggesting a complex role for AXINs.2 Rebouissou, Franconi et al. studied here a new level of sophistication, describing distinct mutations within the CTNNB1 gene itself. They took advantage of a large cohort of annotated HCCs and HCAs (more than 500), but also of some intermediate stages: HCA/HCC borderline tumors and HCC nodules appearing in HCAs. With this extended database, they could hypothesize some chronological sequence in the somatic mutational events arising during the malignant transformation of a benign tumor. Besides the classical exon‐3 deletions or mutations, Zucman‐Rossi's team recently published another "hotspot" of mutations of CTNNB1, located in exons 7‐8.5 It consists in missense mutations within either the fifth (K335) or the sixth (N387) Armadillo repeat. The 12 Armadillo repeats located in β‐catenin protein are known to be responsible for interactions with crucial partners, such as the LEF/TCFs, APC, AXINs, and E‐Cadherin (Fig. 1B). This initial article, together with Rebouissou, Franconi's one, show that these new mutations lead to a weak activation of β‐catenin signaling, as tested in vitro with reporter activity tests, and that these mutations are enriched in HCAs (Fig. 1C). A weak in vivo effect is observed on β‐catenin activity, as evidenced by the low amounts of glutamine synthetase (GS) and LGR5 overexpressions, both being β‐catenin direct targets used as markers of β‐catenin activity. This study, however, does not elucidate how these mutations functionally impact β‐catenin activation. Some proteins are known to interact with the Y333 residue, that is, the metabolic enzyme, PKM2, and the PTK6 kinase,6 but their relationship to the K335/N387 mutation profile remains to be investigated. On another hand, the S45 mutation appeared also to be a weak mutation (i.e., leading to weak levels of β‐catenin‐dependent activities). More precisely, it would rather be considered as a cell‐context‐dependent mutation, the consequences of which differing depending on the cell line studied. It is also the case for the T41 mutation, which has intermediate impact on β‐catenin activity. Only one publication previously suggested that the S45 mutant was not fully resistant to proteolytic degradation.8 This mutation appears at the same frequency in HCAs and in HCCs. But an important and novel result is that, in HCCs, S45 mutation is associated with either a duplication of the mutated allele, an additional mutation in the second allele of CTNNB1, or a monoallelic mutation of APC. These additional events transform a weak mutation into a strong one, through a dramatic reinforcement of β‐catenin activation. In any event, patients with S45‐mutated HCCs have a longer life expectancy than those with other CTNNB1 mutations. Finally, mutations within the DSGIHS box are strong ones, confirming this box as crucial for β‐catenin degradation. They are enriched in malignant lesions. Strong also are the consequences of the either partial or complete loss of the exon 3. Singularly, such deletions are not enriched in HCCs, but are found frequently in borderline lesions. Young patients preferentially carry these deletions in their tumors, and this is reminiscent of HBs, these primary tumors of embryonic or neonatal origin in which deletions of exon 3 are the most frequent CTNNB1 mutational event.9 More knowledge has to be brought concerning the occurrence of these exon 3 deletions and on their maintenance during carcinogenesis. A number of lessons can be learnt from this highly valuable, even if somehow descriptive, study. It appears mandatory that CTNNB1 mutations be systematically assessed in HCAs for two reasons: First, classical GS immunohistochemistry is not sensitive enough to identify S45‐mutated HCAs whereas these HCAs are at risk for malignant transformation; second, HCAs with mutations in the degradation box should be considered at higher risk for an evolution toward HCC. Another message is that two types of mutations are here described, similarly to classical genetic diseases. The strong mutations (within the degradation box) are dominant, given that they seem to be sufficient to drive malignant carcinogenesis. The weak ones affecting S45 residue are recessive, because they need other mutational events to be oncogenic. K335/N387 mutations remain rare in HCCs and are also associated with bi‐allelic mutation or an additional mutation, such as that of TP53, suggesting that these HCA‐associated mutations have only a mild oncogenic impact. Last, this study is provocative for researchers in the Wnt field: The paradigmatic sequence between S45 phosphorylation by casein kinase 1 (CK1) and the subsequent phosphorylation/ubiquitination of adjacent residues by GSK3/β‐TrCP has to be revisited. Another exciting issue for the Wnt researchers is to decipher what are the functional consequences of Y335/N387 mutations. In conclusion, β‐catenin‐mutated HCCs now appear not to be a homogenous entity. Some targeted therapies have been proposed for these cancers.10 These targeted therapies will have to prove their efficiency by also taking into account the location of CTNNB1 mutations. Author names in bold designate shared co‐first authorship.