Innovative mouse models for the tumor suppressor activity of Protocadherin-10 isoforms.

BACKGROUND: Nonclustered mouse protocadherin genes (Pcdh) encode proteins with a typical single ectodomain and a cytoplasmic domain with conserved motifs completely different from those of classic cadherins. Alternative splice isoforms differ in the size of these cytoplasmic domains. In view of the compelling evidence for gene silencing of protocadherins in human tumors, we started investigations on Pcdh functions in mouse cancer models. METHODS: For Pcdh10, we generated two mouse lines: one with floxed exon 1, leading to complete Pcdh10 ablation upon Cre action, and one with floxed exons 2 and 3, leading to ablation of only the long isoforms of Pcdh10. In a mouse medulloblastoma model, we used GFAP-Cre action to locally ablate Pcdh10 in combination with Trp53 and Rb1 ablation. From auricular tumors, that also arose, we obtained tumor-derived cell lines, which were analyzed for malignancy in vitro and in vivo. By lentiviral transduction, we re-expressed Pcdh10 cDNAs. RNA-Seq analyses were performed on these cell families. RESULTS: Surprisingly, not only medulloblastomas were generated in our model but also tumors of tagged auricles (pinnae). For both tumor types, ablation of either all or only long isoforms of Pcdh10 aggravated the disease. We argued that the perichondrial stem cell compartment is at the origin of the pinnal tumors. Immunohistochemical analysis of these tumors revealed different subtypes. We obtained several pinnal-tumor derived (PTD) cell lines and analyzed these for anchorage-independent growth, invasion into collagen matrices, tumorigenicity in athymic mice. Re-expression of either the short or a long isoform of Pcdh10 in two PTD lines counteracted malignancy in all assays. RNA-Seq analyses of these two PTD lines and their respective Pcdh10-rescued cell lines allowed to identify many interesting differentially expressed genes, which were largely different in the two cell families. CONCLUSIONS: A new mouse model was generated allowing for the first time to examine the remarkable tumor suppression activity of protocadherin-10 in vivo. Despite lacking several conserved motifs, the short isoform of Pcdh10 was fully active as tumor suppressor. Our model contributes to scrutinizing the complex molecular mechanisms of tumor initiation and progression upon PCDH10 silencing in many human cancers.

Recurrent chromosomal imbalances provide selective advantage to human embryonic stem cells under enhanced replicative stress conditions.

Human embryonic stem cells (hESCs) and embryonal tumors share a number of common features, including a compromised G1/S checkpoint. Consequently, these rapidly dividing hESCs and cancer cells undergo elevated levels of replicative stress, inducing genomic instability that drives chromosomal imbalances. In this context, it is of interest that long-term in vitro cultured hESCs exhibit a remarkable high incidence of segmental DNA copy number gains, some of which are also highly recurrent in certain malignancies such as 17q gain (17q+). The selective advantage of DNA copy number changes in these cells has been attributed to several underlying processes including enhanced proliferation. We hypothesized that these recurrent chromosomal imbalances become rapidly embedded in the cultured hESCs through a replicative stress driven Darwinian selection process. To this end, we compared the effect of hydroxyurea-induced replicative stress vs normal growth conditions in an equally mixed cell population of isogenic euploid and 17q + hESCs. We could show that 17q + hESCs rapidly overtook normal hESCs. Our data suggest that recurrent chromosomal segmental gains provide a proliferative advantage to hESCs under increased replicative stress, a process that may also explain the highly recurrent nature of certain imbalances in cancer.

Neural defects caused by total and Wnt1-Cre mediated ablation of p120ctn in mice.

BACKGROUND: p120 catenin (p120ctn) is an important component in the cadherin-catenin cell adhesion complex because it stabilizes cadherin-mediated intercellular junctions. Outside these junctions, p120ctn is actively involved in the regulation of small GTPases of the Rho family, in actomyosin dynamics and in transcription regulation. We and others reported that loss of p120ctn in mouse embryos results in an embryonic lethal phenotype, but the exact developmental role of p120ctn during brain formation has not been reported. RESULTS: We combined floxed p120ctn mice with Del-Cre or Wnt1-Cre mice to deplete p120ctn from either all cells or specific brain and neural crest cells. Complete loss of p120ctn in mid-gestation embryos resulted in an aberrant morphology, including growth retardation, failure to switch from lordotic to fetal posture, and defective neural tube formation and neurogenesis. By expressing a wild-type p120ctn from the ROSA26 locus in p120ctn-null mouse embryonic stem cells, we could partially rescue neurogenesis. To further investigate the developmental role of p120ctn in neural tube formation, we generated conditional p120ctnfl/fl;Wnt1Cre knockout mice. p120ctn deletion in Wnt1-expressing cells resulted in neural tube closure defects (NTDs) and craniofacial abnormalities. These defects could not be correlated with misregulation of brain marker genes or cell proliferation. In contrast, we found that p120ctn is required for proper expression of the cell adhesion components N-cadherin, E-cadherin and ß-catenin, and of actin-binding proteins cortactin and Shroom3 at the apical side of neural folds. This region is of critical importance for closure of neural folds. Surprisingly, the lateral side of mutant neural folds showed loss of p120ctn, but not of N-cadherin, ß-catenin or cortactin. CONCLUSIONS: These results indicate that p120ctn is required for neurogenesis and neurulation. Elimination of p120ctn in cells expressing Wnt1 affects neural tube closure by hampering correct formation of specific adhesion and actomyosin complexes at the apical side of neural folds. Collectively, our results demonstrate the crucial role of p120ctn during brain morphogenesis.

Targeted AURKA degradation: Towards new therapeutic agents for neuroblastoma.

Aurora kinase A (AURKA) is a well-established target in neuroblastoma (NB) due to both its catalytic functions during mitosis and its kinase-independent functions, including stabilization of the key oncoprotein MYCN. We present a structure-activity relationship (SAR) study of MK-5108-derived PROTACs against AURKA by exploring different linker lengths and exit vectors on the thalidomide moiety. PROTAC SK2188 induces the most potent AURKA degradation (DC50,24h 3.9 nM, Dmax,24h 89%) and shows an excellent binding and degradation selectivity profile. Treatment of NGP neuroblastoma cells with SK2188 induced concomitant MYCN degradation, high replication stress/DNA damage levels and apoptosis. Moreover, SK2188 significantly outperforms the parent inhibitor MK-5108 in a cell proliferation screen and patient-derived organoids. Furthermore, altering the attachment point of the PEG linker to the 5-position of thalidomide allowed us to identify a potent AURKA degrader with a linker as short as 2 PEG units. With this, our SAR-study provides interesting lead structures for further optimization and validation of AURKA degradation as a potential therapeutic strategy in neuroblastoma.

SOX11 regulates SWI/SNF complex components as member of the adrenergic neuroblastoma core regulatory circuitry.

The pediatric extra-cranial tumor neuroblastoma displays a low mutational burden while recurrent copy number alterations are present in most high-risk cases. Here, we identify SOX11 as a dependency transcription factor in adrenergic neuroblastoma based on recurrent chromosome 2p focal gains and amplifications, specific expression in the normal sympatho-adrenal lineage and adrenergic neuroblastoma, regulation by multiple adrenergic specific (super-)enhancers and strong dependency on high SOX11 expression in adrenergic neuroblastomas. SOX11 regulated direct targets include genes implicated in epigenetic control, cytoskeleton and neurodevelopment. Most notably, SOX11 controls chromatin regulatory complexes, including 10 SWI/SNF core components among which SMARCC1, SMARCA4/BRG1 and ARID1A. Additionally, the histone deacetylase HDAC2, PRC1 complex component CBX2, chromatin-modifying enzyme KDM1A/LSD1 and pioneer factor c-MYB are regulated by SOX11. Finally, SOX11 is identified as a core transcription factor of the core regulatory circuitry (CRC) in adrenergic high-risk neuroblastoma with a potential role as epigenetic master regulator upstream of the CRC.

RRM2 enhances MYCN-driven neuroblastoma formation and acts as a synergistic target with CHK1 inhibition.

High-risk neuroblastoma, a pediatric tumor originating from the sympathetic nervous system, has a low mutation load but highly recurrent somatic DNA copy number variants. Previously, segmental gains and/or amplifications allowed identification of drivers for neuroblastoma development. Using this approach, combined with gene dosage impact on expression and survival, we identified ribonucleotide reductase subunit M2 (RRM2) as a candidate dependency factor further supported by growth inhibition upon in vitro knockdown and accelerated tumor formation in a neuroblastoma zebrafish model coexpressing human RRM2 with MYCN. Forced RRM2 induction alleviates excessive replicative stress induced by CHK1 inhibition, while high RRM2 expression in human neuroblastomas correlates with high CHK1 activity. MYCN-driven zebrafish tumors with RRM2 co-overexpression exhibit differentially expressed DNA repair genes in keeping with enhanced ATR-CHK1 signaling activity. In vitro, RRM2 inhibition enhances intrinsic replication stress checkpoint addiction. Last, combinatorial RRM2-CHK1 inhibition acts synergistic in high-risk neuroblastoma cell lines and patient-derived xenograft models, illustrating the therapeutic potential.

Zebrafish teeth as a model for repetitive epithelial morphogenesis: dynamics of E-cadherin expression.

BACKGROUND: The development of teeth is the result of interactions between competent mesenchyme and epithelium, both of which undergo extensive morphogenesis. The importance of cell adhesion molecules in morphogenesis has long been acknowledged but remarkably few studies have focused on the distribution and function of these molecules in tooth development. RESULTS: We analyzed the expression pattern of an important epithelial cadherin, E-cadherin, during the formation of first-generation teeth as well as replacement teeth in the zebrafish, using in situ hybridization and whole mount immunostaining to reveal mRNA expression and protein distribution. E-cadherin was detected in every layer of the enamel organ during the different stages of tooth development, but there were slight differences between first-generation and replacement teeth in the strength and distribution of the signal. The dental papilla, which is derived from the mesenchyme, did not show any expression. Remarkably, the crypts surrounding the functional teeth showed an uneven distribution of E-cadherin throughout the pharyngeal region. CONCLUSIONS: The slight differences between E-cadherin expression in zebrafish teeth and developing mouse and human teeth are discussed in the light of fundamental differences in structural and developmental features of the dentition between zebrafish and mammals. Importantly, the uninterrupted expression of E-cadherin indicates that down-regulation of E-cadherin is not required for formation of an epithelial tooth bud. Further research is needed to understand the role of other cell adhesion systems during the development of teeth and the formation of replacement teeth.

Armc8 is an evolutionarily conserved armadillo protein involved in cell-cell adhesion complexes through multiple molecular interactions.

Armadillo-repeat-containing protein 8 (Armc8) belongs to the family of armadillo-repeat containing proteins, which have been found to be involved in diverse cellular functions including cell-cell contacts and intracellular signaling. By comparative analyses of armadillo repeat protein structures and genomes from various premetazoan and metazoan species, we identified orthologs of human Armc8 and analyzed in detail the evolutionary relationship of Armc8 genes and their encoded proteins. Armc8 is a highly ancestral armadillo protein although not present in yeast. Consequently, Armc8 is not the human ortholog of yeast Gid5/Vid28.Further, we performed a candidate approach to characterize new protein interactors of Armc8. Interactions between Armc8 and specific δ-catenins (plakophilins-1, -2, -3 and p0071) were observed by the yeast two-hybrid approach and confirmed by co-immunoprecipitation and co-localization. We also showed that Armc8 interacts specifically with αE-catenin but neither with αN-catenin nor with αT-catenin. Degradation of αE-catenin has been reported to be important in cancer and to be regulated by Armc8. A similar process may occur with respect to plakophilins in desmosomes. Deregulation of desmosomal proteins has been considered to contribute to tumorigenesis.

GC Content of Early Metazoan Genes and Its Impact on Gene Expression Levels in Mammalian Cell Lines.

With the genomes available for many animal clades, including the early-branching metazoans, one can readily study the functional conservation of genes across a diversity of animal lineages. Ectopic expression of an animal protein in, for instance, a mammalian cell line is a generally used strategy in structure-function analysis. However, this might turn out to be problematic in case of distantly related species. Here we analyzed the GC content of the coding sequences of basal animals and show its impact on gene expression levels in human cell lines, and, importantly, how this expression efficiency can be improved. Optimization of the GC3 content in the coding sequences of cadherin, alpha-catenin, and paracaspase of Trichoplax adhaerens dramatically increased the expression of these basal animal genes in human cell lines.

TALEN-mediated apc mutation in Xenopus tropicalis phenocopies familial adenomatous polyposis.

Truncating mutations in the tumor suppressor gene adenomatous polyposis coli (APC) are the initiating step in the vast majority of sporadic colorectal cancers, and they underlie familial adenomatous polyposis (FAP) syndromes. Modeling of APC- driven tumor formation in the mouse has contributed substantially to our mechanistic understanding of the associated disease, but additional models are needed to explore therapeutic opportunities and overcome current limitations of mouse models. We report on a novel and penetrant genetic cancer model in Xenopus tropicalis, an aquatic tetrapod vertebrate with external development, diploid genome and short life cycle. Tadpoles and froglets derived from embryos injected with TAL effector nucleases targeting the apc gene rapidly developed intestinal hyperplasia and other neoplasms observed in FAP patients, including desmoid tumors and medulloblastomas. Bi-allelic apc mutations causing frame shifts were detected in the tumors, which displayed activation of the Wnt/ß-catenin pathway and showed increased cellular proliferation. We further demonstrate that simultaneous double bi-allelic mutation of apc and a non-relevant gene is possible in the neoplasias, opening the door for identification and characterization of effector or modifier genes in tumors expressing truncated apc. Our results demonstrate the power of modeling human cancer in Xenopus tropicalis using mosaic TALEN-mediated bi-allelic gene disruption.

Inter- and intrafamilial variability in premature pubarche and polycystic ovary syndrome.

OBJECTIVE: To ascertain the extent of phenotypic heterogeneity for premature pubarche and polycystic ovary syndrome between and within families. DESIGN: Association study. SETTING: Academic research environment. PATIENT(S): Three families in which the propositus had presented with either premature pubic hair or adolescent hyperandrogenism. INTERVENTION(S): Detailed medical histories, hormone determinations, and genotype analyses. MAIN OUTCOME MEASURE(S): Clinical phenotype. Genotypes for CYP21, HSD3B2, G972R variant of IRS-1, N363S variant of GRL, W64R variant of ADRB3, CAG repeat in exon 1 of AR, MspAI polymorphism in CYP17, and R264C variant of CYP19. RESULT(S): Significant phenotypic and genetic heterogeneity was observed both within and between families. In one family, CYP21 and IRS-1 variants were observed to co-segregate with symptoms of androgen excess and obesity. No genetic markers were consistently noted to associate with clinical features of hyperandrogenism in the other two families. CONCLUSION(S): The difficulties in classifying female family members as clearly affected or unaffected and lack of definitive male phenotype complicate the use of linkage analysis to identify the polycystic ovary syndrome genes. Each family is best considered on an individual basis to identify genetic markers that segregate with the clinical features of androgen excess.

Whole mount immunohistochemistry and in situ hybridization of larval and adult zebrafish dental tissues.

Tooth development is increasingly being studied in a variety of vertebrate model organisms, each contributing its own perspective to our understanding of dental diversity. In situ hybridization and immunohistochemistry are well-established and frequently used techniques to study the presence of mRNA and protein. Here, we describe a protocol for whole mount immunohistochemistry and in situ hybridization that can be applied to all stages of zebrafish development and dissected bony parts. The description of these protocols is followed by the outline of a quick decalcification method and the procedure for embedding in epoxy resin to obtain serial sections with high histological quality.

Ocular anterior segment dysgenesis upon ablation of p120 catenin in neural crest cells.

PURPOSE: Development of the ocular anterior segment depends largely on periocular mesenchyme cells, which are derived predominantly from neural crest cells (NCC). Specific and differential cell adhesion is expected to be instrumental in induction, migration, and differentiation of NCC. As p120 catenin (ctn) is an important component of cadherin-catenin cell adhesion complexes, we assessed its role in development of the anterior segment structure. METHODS: We generated conditional p120ctn(fl/fl);Wnt1Cre knockout mice and studied the effect of this gene ablation on eye development in vivo. In addition, p120ctn was knocked down in vitro. RESULTS: Wnt1Cre-mediated deletion of floxed p120ctn alleles in NCC resulted in serious ocular anterior segment dysgenesis (ASD), including iridocorneal angle closure, complete anterior chamber obliteration, iris and ciliary body hypoplasia, corneal malformation and opacity, and glaucoma-like defects. A completely penetrant phenotype was visible approximately three weeks after birth, but histologic defects were obvious at embryonal day 18.5 (E18.5). Neither migration of NCC nor expression of key transcription factors appeared to be affected. In contrast, the N-cadherin expression pattern was changed significantly in iridocorneal angle cells and corneal endothelium. A human trabecular meshwork cell line in which p120ctn was knocked down also showed decreased expression levels of N-cadherin and ß-catenin at the plasma membrane, but no defect in cell migration. CONCLUSIONS: p120ctn has a critical role in ocular mesenchyme development. Loss of p120ctn and the associated N-cadherin downregulation in NCC leads to ASD without affecting cell migration. p120ctn abnormalities might have a role in the pathophysiology of mammalian eye development.