Claudio Cantù

Persistent Wnt/β-Catenin Signaling Determines Dorsalization of the Postnatal Subventricular Zone and Neural Stem Cell Specification into Oligodendrocytes and Glutamatergic Neurons

In the postnatal and adult central nervous system (CNS), the subventricular zone (SVZ) of the forebrain is the main source of neural stem cells (NSCs) that generate olfactory neurons and oligodendrocytes (OLs), the myelinating cells of the CNS. Here, we provide evidence of a primary role for canonical Wnt/β‐catenin signaling in regulating NSC fate along neuronal and oligodendroglial lineages in the postnatal SVZ. Our findings demonstrate that glutamatergic neuronal precursors (NPs) and oligodendrocyte precursors (OPs) are derived strictly from the dorsal SVZ (dSVZ) microdomain under the control of Wnt/β‐catenin, whereas GABAergic NPs are derived mainly from the lateral SVZ (lSVZ) microdomain independent of Wnt/β‐catenin. Transcript analysis of microdissected SVZ microdomains revealed that canonical Wnt/β‐catenin signaling was more pronounced in the dSVZ microdomain. This was confirmed using the β‐catenin‐activated Wnt‐reporter mouse and by pharmacological stimulation of Wnt/β‐catenin by infusion of the specific glycogen synthase kinase 3β inhibitor, AR‐A014418, which profoundly increased the generation of cycling cells. In vivo genetic/pharmacological stimulation or inhibition of Wnt/β‐catenin, respectively, increased and decreased the differentiation of dSVZ‐NSCs into glutamatergic NPs, and had a converse effect on GABAergic NPs. Activation of Wnt/β‐catenin dramatically stimulated the generation of OPs, but its inhibition had no effect, indicating other factors act in concert with Wnt/β‐catenin to fine tune oligodendrogliogenesis in the postnatal dSVZ. These results demonstrate a role for Wnt/β‐catenin signaling within the dorsal microdomain of the postnatal SVZ, in regulating the genesis of glutamatergic neurons and OLs. Stem Cells 2014;32:1301–1312

Wnt/β‐Catenin Signaling Regulates Sequential Fate Decisions of Murine Cortical Precursor Cells

The fate of neural progenitor cells (NPCs) is determined by a complex interplay of intrinsic programs and extrinsic signals, very few of which are known. β‐Catenin transduces extracellular Wnt signals, but also maintains adherens junctions integrity. Here, we identify for the first time the contribution of β‐catenin transcriptional activity as opposed to its adhesion role in the development of the cerebral cortex by combining a novel β‐catenin mutant allele with conditional inactivation approaches. Wnt/β‐catenin signaling ablation leads to premature NPC differentiation, but, in addition, to a change in progenitor cell cycle kinetics and an increase in basally dividing progenitors. Interestingly, Wnt/β‐catenin signaling affects the sequential fate switch of progenitors, leading to a shortened neurogenic period with decreased number of both deep and upper‐layer neurons and later, to precocious astrogenesis. Indeed, a genome‐wide analysis highlighted the premature activation of a corticogenesis differentiation program in the Wnt/β‐catenin signaling‐ablated cortex. Thus, β‐catenin signaling controls the expression of a set of genes that appear to act downstream of canonical Wnt signaling to regulate the stage‐specific production of appropriate progenitor numbers, neuronal subpopulations, and astroglia in the forebrain. Stem Cells 2015;33:170–182

BCL9/9L-β-catenin Signaling is Associated With Poor Outcome in Colorectal Cancer

BCL9/9L proteins enhance the transcriptional output of the β-catenin/TCF transcriptional complex and contribute critically to upholding the high WNT signaling level required for stemness maintenance in the intestinal epithelium. Here we show that a BCL9/9L-dependent gene signature derived from independent mouse colorectal cancer (CRC) models unprecedentedly separates patient subgroups with regard to progression free and overall survival. We found that this effect was by and large attributable to stemness related gene sets. Remarkably, this signature proved associated with recently described poor prognosis CRC subtypes exhibiting high stemness and/or epithelial-to-mesenchymal transition (EMT) traits. Consistent with the notion that high WNT signaling is required for stemness maintenance, ablating Bcl9/9l-β-catenin in murine oncogenic intestinal organoids provoked their differentiation and completely abrogated their tumorigenicity, while not affecting their proliferation. Therapeutic strategies aimed at targeting WNT responses may be limited by intestinal toxicity. Our findings suggest that attenuating WNT signaling to an extent that affects stemness maintenance without disturbing intestinal renewal might be well tolerated and prove sufficient to reduce CRC recurrence and dramatically improve disease outcome.

Pharmacogenomic identification of small molecules for lineage specific manipulation of subventricular zone germinal activity

Strategies for promoting neural regeneration are hindered by the difficulty of manipulating desired neural fates in the brain without complex genetic methods. The subventricular zone (SVZ) is the largest germinal zone of the forebrain and is responsible for the lifelong generation of interneuron subtypes and oligodendrocytes. Here, we have performed a bioinformatics analysis of the transcriptome of dorsal and lateral SVZ in early postnatal mice, including neural stem cells (NSCs) and their immediate progenies, which generate distinct neural lineages. We identified multiple signaling pathways that trigger distinct downstream transcriptional networks to regulate the diversity of neural cells originating from the SVZ. Next, we used a novel in silico genomic analysis, searchable platform-independent expression database/connectivity map (SPIED/CMAP), to generate a catalogue of small molecules that can be used to manipulate SVZ microdomain-specific lineages. Finally, we demonstrate that compounds identified in this analysis promote the generation of specific cell lineages from NSCs in vivo, during postnatal life and adulthood, as well as in regenerative contexts. This study unravels new strategies for using small bioactive molecules to direct germinal activity in the SVZ, which has therapeutic potential in neurodegenerative diseases.

The Pygo2-H3K4me2/3 interaction is dispensable for mouse development and Wnt signaling-dependent transcription

Pygopus has been discovered as a fundamental Wnt signaling component in Drosophila. The mouse genome encodes two Pygopus homologs, Pygo1 and Pygo2. They serve as context-dependent β-catenin coactivators, with Pygo2 playing the more important role. All Pygo proteins share a highly conserved plant homology domain (PHD) that allows them to bind di- and trimethylated lysine 4 of histone H3 (H3K4me2/3). Despite the structural conservation of this domain, the relevance of histone binding for the role of Pygo2 as a Wnt signaling component and as a reader of chromatin modifications remains speculative. Here we generate a knock-in mouse line, homozygous for a Pygo2 mutant defective in chromatin binding. We show that even in the absence of the potentially redundant Pygo1, Pygo2 does not require the H3K4me2/3 binding activity to sustain its function during mouse development. Indeed, during tissue homeostasis, Wnt/β-catenin-dependent transcription is largely unaffected. However, the Pygo2-chromatin interaction is relevant in testes, where, importantly, Pygo2 binds in vivo to the chromatin in a PHD-dependent manner. Its presence on regulatory regions does not affect the transcription of nearby genes; rather, it is important for the recruitment of the histone acetyltransferase Gcn5 to chromatin, consistent with a testis-specific and Wnt-unrelated role for Pygo2 as a chromatin remodeler.

Pharmacological interventions in the Wnt pathway: inhibition of Wnt secretion versus disrupting the protein–protein interfaces of nuclear factors

Mutations in components of the Wnt pathways are a frequent cause of many human diseases, particularly cancer. Despite the fact that a causative link between aberrant Wnt signalling and many types of human cancers was established more than a decade ago, no Wnt signalling inhibitors have made it into the clinic so far. One reason for this is that no pathway‐specific kinase is known. Additionally, targeting the protein–protein interactions needed to transduce the signal has not met with success so far. Complicating the search for and use of inhibitors is the complexity of the cascades triggered by the Wnts and their paramount biological importance. Wnt/β‐catenin signalling is involved in virtually all aspects of embryonic development and in the control of the homeostasis of adult tissues. Encouragingly, however, in recent years, first successes with Wnt‐pathway inhibitors have been reported in mouse models of disease. In this review, we summarize possible roads to follow during the quest to pharmacologically modulate the Wnt signalling pathway in cancer.

A cytoplasmic role of Wnt/β-catenin transcriptional cofactors Bcl9, Bcl9l, and Pygopus in tooth enamel formation

Transcriptional cofactors that participate in Wnt/β-catenin signaling act in the cytoplasm to promote proper development of tooth enamel. Cytoplasmic functions for transcriptional cofactors in teeth Bcl9, Bcl9l, and Pygo2 interact with transcription factors, such as the Wnt-regulated protein β-catenin, to regulate gene expression. Cantù et al. reveal that these proteins also have cytoplasmic functions during tooth development and are particularly important for the formation of enamel. Mice lacking both Pygo1 and Pygo2 or both Bcl9 and Bcl9l developed teeth, a process that requires Wnt/β-catenin transcriptional regulation, but the enamel was structurally disorganized and contained less iron than teeth from control mice. Bcl9, Bcl9l, and Pygo2 were present in the cytoplasm of ameloblasts, the cells that secrete enamel proteins, and colocalized in these cells with amelogenin, the main component of enamel. Bcl9 interacted with amelogenin and proteins involved in exocytosis and vesicular trafficking, suggesting that these proteins function in the trafficking or secretion of enamel proteins. These results demonstrate that Bcl9, Bcl9l, and Pygo2 have cytoplasmic functions distinct from their roles as transcriptional cofactors downstream of Wnt signaling. Wnt-stimulated β-catenin transcriptional regulation is necessary for the development of most organs, including teeth. Bcl9 and Bcl9l are tissue-specific transcriptional cofactors that cooperate with β-catenin. In the nucleus, Bcl9 and Bcl9l simultaneously bind β-catenin and the transcriptional activator Pygo2 to promote the transcription of a subset of Wnt target genes. We showed that Bcl9 and Bcl9l function in the cytoplasm during tooth enamel formation in a manner that is independent of Wnt-stimulated β-catenin–dependent transcription. Bcl9, Bcl9l, and Pygo2 localized mainly to the cytoplasm of the epithelial-derived ameloblasts, the cells responsible for enamel production. In ameloblasts, Bcl9 interacted with proteins involved in enamel formation and proteins involved in exocytosis and vesicular trafficking. Conditional deletion of both Bcl9 and Bcl9l or both Pygo1 and Pygo2 in mice produced teeth with defective enamel that was bright white and deficient in iron, which is reminiscent of human tooth enamel pathologies. Overall, our data revealed that these proteins, originally defined through their function as β-catenin transcriptional cofactors, function in odontogenesis through a previously uncharacterized cytoplasmic mechanism, revealing that they have roles beyond that of transcriptional cofactors.

Linking dental pathologies and cancer via Wnt signalling

Great efforts have been made over the past decades to discover new therapeutic targets for a big variety of human pathologies. Most of the studies dealing with severe pathological conditions such as cancers and tissue malformations are focused on the role of either widely recognized master controlling genes such as ras and myc or pivotal components of key signalling pathways, among which Wnt and Notch [1, 2]. However, these genes and molecules are fundamental for proper embryogenesis as well as for tissue and organ homeostasis and regeneration, where they regulate cell proliferation, migration, differentiation and apoptosis. The precise timing and localization of their activation are important to ensure the appropriate cellular functions in physiological conditions. Thus, their indiscriminate targeting is not desirable, due to both the high risk of severe side effects and the certainty of broad phenotypic consequences. On the contrary, optimal therapeutic targets should be selected based on their tissue, time and pathology specific roles. Here, we suggest a paradigmatic example of such target molecules that could be represented by the Wnt/β-catenin signalling components Bcl9 and Bcl9l.

A RING finger to wed TCF and β-catenin

In this issue of EMBO reports, Waterman and colleagues identify RNF14 as a new enhancer of the Wnt-dependent transcriptional outputs that acts at the level of the TCF/LEF–β-catenin complex.

Pharmacophore-guided discovery of CDC25 inhibitors causing cell cycle arrest and cell death

CDC25 phosphatases have a key role in cell cycle transitions and are important targets for cancer therapy. Here, we set out to discover novel CDC25 inhibitors. Using a combination of computational approaches we defined a minimal common pharmacophore in established CDC25 inhibitors and performed a virtual screening of a proprietary library. Taking advantage of the availability of crystal structures for CDC25A and CDC25B and using a molecular docking strategy, we carried out hit expansion/optimization. Enzymatic assays revealed that naphthoquinone scaffolds were the most promising CDC25 inhibitors among selected hits. At the molecular level, the compounds acted through a mixed-type mechanism of inhibition of phosphatase activity, involving reversible oxidation of cysteine residues. In 2D cell cultures, the compounds caused arrest of the cell cycle at the G1/S or at the G2/M transition. Mitotic markers analysis and time-lapse microscopy confirmed that CDK1 activity was impaired and that mitotic arrest was followed by death. Finally, studies on 3D organoids derived from intestinal crypt stem cells of Apc/K-Ras mice revealed that the compounds caused arrest of proliferation.