New insights into diagnosis and therapeutic options for proliferative hepatoblastoma.

Surgery and cisplatin-based treatment of hepatoblastoma (HB) currently guarantee the survival of 70%-80% of patients. However, some important challenges remain in diagnosing high-risk tumors and identifying relevant targetable pathways offering new therapeutic avenues. Previously, two molecular subclasses of HB tumors have been described, C1 and C2, with C2 being the subgroup with the poorest prognosis, a more advanced tumor stage, and the worst overall survival rate. An associated 16-gene signature to discriminate the two tumoral subgroups was proposed, but it has not been transferred into clinical routine. To address these issues, we performed RNA sequencing of 25 tumors and matched normal liver samples from patients. The transcript profiling separated HB into three distinct subgroups named C1, C2A, and C2B, identifiable by a concise four-gene signature: hydroxysteroid 17-beta dehydrogenase 6, integrin alpha 6, topoisomerase 2-alpha, and vimentin, with topoisomerase 2-alpha being characteristic for the proliferative C2A tumors. Differential expression of these genes was confirmed by quantitative RT-PCR on an expanded cohort and by immunohistochemistry. We also revealed significant overexpression of genes involved in the Fanconi anemia (FA) pathway in the C2A subgroup. We then investigated the ability of several described FA inhibitors to block growth of HB cells in vitro and in vivo. We demonstrated that bortezomib, a Food and Drug Administration-approved proteasome inhibitor, strongly impairs the proliferation and survival of HB cell lines in vitro, blocks FA pathway-associated double-strand DNA repair, and significantly impedes HB growth in vivo. CONCLUSION: The highly proliferating C2A subtype is characterized by topoisomerase 2-alpha gene up-regulation and FA pathway activation, and the HB therapeutic arsenal could include bortezomib for the treatment of patients with the most aggressive tumors. (Hepatology 2018;68:89-102).

New tumor suppressor microRNAs target glypican-3 in human liver cancer.

Glypican-3 (GPC3) is an oncogene, frequently upregulated in liver malignancies such as hepatocellular carcinoma (HCC) and hepatoblastoma and constitutes a potential molecular target for therapy in liver cancer. Using a functional screening system, we identified 10 new microRNAs controlling GPC3 expression in malignant liver cells, five of them e.g. miR-4510, miR-203a-3p, miR-548aa, miR-376b-3p and miR-548v reduce GPC3 expression. These 5 microRNAs were significantly downregulated in tumoral compared to non-tumoral liver and inhibited tumor cell proliferation. Interestingly, miR-4510 inversely correlated with GPC3 mRNA and protein in HCC samples. This microRNA also induced apoptosis of hepatoma cells and blocked tumor growth in vivo in the chick chorioallantoic membrane model. We further show that the tumor suppressive effect of miR-4510 is mediated through direct targeting of GPC3 mRNA and inactivation of Wnt/ß-catenin transcriptional activity and signaling pathway. Moreover, miR-4510 up-regulated the expression of several tumor suppressor genes while reducing the expression of other pro-oncogenes. In summary, we uncovered several new microRNAs targeting the oncogenic functions of GPC3. We provided strong molecular, cellular and in vivo evidences for the tumor suppressive activities of miR-4510 bringing to the fore the potential value of this microRNA in HCC therapy.

MicroRNA therapy inhibits hepatoblastoma growth in vivo by targeting ß-catenin and Wnt signaling.

Hepatoblastoma (HBL) is the most common pediatric liver cancer. In this malignant neoplasm, beta-catenin protein accumulates and increases Wnt signaling due to recurrent activating mutations in the catenin-beta 1 (CTNNB1) gene. Therefore, beta-catenin is a key therapeutic target in HBL. However, controlling beta-catenin production with therapeutic molecules has been challenging. New biological studies could provide alternative therapeutic solutions for the treatment of HBL, especially for advanced tumors and metastatic disease. In this study, we identified microRNAs (miRNAs) that target beta-catenin and block HBL cell proliferation in vitro and tumor growth in vivo. Using our dual-fluorescence-FunREG system, we screened a library of 1,712 miRNA mimics and selected candidates inhibiting CTNNB1 expression through interaction with its untranslated regions. After validating the regulatory effect of nine miRNAs on beta-catenin in HBL cells, we measured their expression in patient samples. Let-7i-3p, miR-449b-3p, miR-624-5p, and miR-885-5p were decreased in tumors compared to normal livers. Moreover, they inhibited HBL cell growth and Wnt signaling activity in vitro partly through beta-catenin down-regulation. Additionally, miR-624-5p induced cell senescence in vitro, blocked experimental HBL growth in vivo, and directly targeted the beta-catenin 3'-untranslated region. Conclusion: Our results shed light on how beta-catenin-regulating miRNAs control HBL progression through Wnt signaling inactivation. In particular, miR-624-5p may constitute a promising candidate for miRNA replacement therapy for HBL patients. (Hepatology Communications 2017;1:168-183).

Conditional ablation of NKp46+ cells using a novel Ncr1(greenCre) mouse strain: NK cells are essential for protection against pulmonary B16 metastases.

To study gene functions specifically in NKp46+ cells we developed novel Cre mice allowing for conditional gene targeting in cells expressing Ncr1 (encoding NKp46). We generated transgenic Ncr1(greenCre) mice carrying an EGFPcre fusion under the control of a proximal Ncr1 promoter that faithfully directed EGFPcre expression to NKp46+ cells from lymphoid and nonlymphoid tissues. This approach allowed for direct detection of Cre-expressing NKp46+ cells via their GFP signature by flow cytometry and histology. Cre was functional as evidenced by the NKp46+ cell-specific expression of RFP in Ncr1(greenCre) Rosa-dtRFP reporter mice. We generated Ncr1(greenCre) Il2rg(fl/fl) mice that lack NKp46+ cells in an otherwise intact hematopoietic environment. Il2rg encodes the common gamma chain (γc ), which is an essential receptor subunit for cytokines (IL-2, -4, -7, -9, -15, and -21) that stimulate lymphocyte development and function. In Ncr1(greenCre) Il2rg(fl/fl) mice, NK cells are severely reduced and the few remaining NKp46+ cells escaping γc deletion failed to express GFP. Using this new NK-cell-deficient model, we demonstrate that the homeostasis of NKp46+ cells from all tissues (including the recently described intraepithelial ILC1 subset) requires Il2rg. Finally, Ncr1(greenCre) Il2rg(fl/fl) mice are unable to reject B16 lung metastases demonstrating the essential role of NKp46+ cells in antimelanoma immune responses.

Proposal for standardization of optimized mycobacterial interspersed repetitive unit-variable-number tandem repeat typing of Mycobacterium tuberculosis.

Molecular typing based on 12 loci containing variable numbers of tandem repeats of mycobacterial interspersed repetitive units (MIRU-VNTRs) has been adopted in combination with spoligotyping as the basis for large-scale, high-throughput genotyping of Mycobacterium tuberculosis. However, even the combination of these two methods is still less discriminatory than IS6110 fingerprinting. Here, we define an optimized set of MIRU-VNTR loci with a significantly higher discriminatory power. The resolution and the stability/robustness of 29 loci were analyzed, using a total of 824 tubercle bacillus isolates, including representatives of the main lineages identified worldwide so far. Five loci were excluded for lack of robustness and/or stability in serial isolates or isolates from epidemiologically linked patients. The use of the 24 remaining loci increased the number of types by 40%--and by 23% in combination with spoligotyping--among isolates from cosmopolitan origins, compared to those obtained with the original set of 12 loci. Consequently, the clustering rate was decreased by fourfold--by threefold in combination with spoligotyping--under the same conditions. A discriminatory subset of 15 loci with the highest evolutionary rates was then defined that concentrated 96% of the total resolution obtained with the full 24-locus set. Its predictive value for evaluating M. tuberculosis transmission was found to be equal to that of IS6110 restriction fragment length polymorphism typing, as shown in a companion population-based study. This 15-locus system is therefore proposed as the new standard for routine epidemiological discrimination of M. tuberculosis isolates and the 24-locus system as a high-resolution tool for phylogenetic studies.

Genetic diversity and population structure of Mycobacterium tuberculosis in Casablanca, a Moroccan city with high incidence of tuberculosis.

Although lower-resource countries have by far the highest burden of tuberculosis, knowledge of Mycobacterium tuberculosis population structure and genetic diversity in these regions remains almost nonexistent. In this paper, 150 Moroccan M. tuberculosis isolates circulating in Casablanca were genotyped by random amplified polymorphic DNA analysis using 10 different primers and by mycobacterial interspersed repetitive units-variable number of tandem repeats typing at 12 loci. The population genetic tests revealed a basically clonal structure for this population, without excluding rare genetic exchanges. Genetic analysis also showed a notable genetic polymorphism for the species M. tuberculosis, a weak cluster individualization, and an unexpected genetic diversity for a population in such a high-incidence community. Phylogenetic analyses of this Moroccan sample also supported that these isolates are genetically heterogeneous.

Genotyping of the Mycobacterium tuberculosis complex using MIRUs: association with VNTR and spoligotyping for molecular epidemiology and evolutionary genetics.

The recent introduction of molecular methods has gained increased acceptance as a powerful tool for epidemiology and phylogeny of tuberculosis (TB). In this investigation, the efficiency of molecular typing using mycobacterial interspersed repetitive units (MIRUs) was assessed on a set of 116 Mycobacterium tuberculosis complex clinical isolates from 11 different geographic origins. The results obtained were compared with spoligotyping and variable number of tandem DNA repeats (VNTRs) typing data. Eighty-nine different MIRU profiles were obtained on the sample studied. Spoligotyping- or VNTR-defined clusters were split into subclusters by MIRU typing. Conversely, almost all of the clinical isolates clustered by MIRUs were shown to belong to spoligotyping-based defined clusters. The calculation of the discriminative power by the Hunter-Gaston index (HGI) for VNTR, spoligotyping and MIRU typing gave the values of, respectively, 0.959, 0.965 and 0.988, showing the high discriminative power of the MIRUs. The allelic diversity of the sample was calculated for each of the MIRU-VNTR loci; five MIRU loci (MIRU nos. 10, 23, 26, 31 and 40) were "highly discriminant", four (MIRU nos. 4, 16, 24 and 39) were "moderately discriminant", and three (MIRU nos. 2, 20 and 27) were "poorly discriminant". Among the three complementary VNTRs (exact tandem repeats ETR-A, ETR-B and ETR-C), ETR-A was the most discriminant locus. A combined numerical analysis of spoligotyping, VNTR and MIRU typing results partly corroborated a recently hypothesized evolutionary scenario for the M. tuberculosis complex. M. canettii would be the first branch to have diverged from a common M. tuberculosis complex ancestor. The East-African Indian (EAI) clade could be the first family to have diverged thereafter. A third branching separated a M. africanum-M. bovis clade, followed by a node separating Beijing versus non-Beijing M. tuberculosis. The Beijing clade was distinct from the Central Asian 1 (CAS1) family. Among non-Beijing strains, branches such as the Latin-American and Mediterranean (LAM), X and Haarlem clades diverged later. In conclusion, the results obtained show the congruence between clades defined by spoligotyping, and MIRU-VNTR, and underline the potential of these methods for M. tuberculosis phylogeny reconstruction. We also conclude that MIRU typing is a very promising method that may be used in a "two PCR-based" genotyping strategy, in conjunction to conventional epidemiological investigations.

Overrepresentation of a gene family encoding extracytoplasmic solute receptors in Bordetella.

A family of genes that are likely to encode extracytoplasmic solute receptors is strongly overrepresented in several beta-proteobacteria, including Bordetella pertussis. This gene family, of which members have been called bug genes, contains some examples that are contained within polycistronic operons coding for tripartite uptake transporters of the TTT family, while the vast majority are "orphan" genes. Proteomic and functional analyses demonstrated that several of these genes are expressed in B. pertussis, and one is involved in citrate uptake. The bug genes probably form an ancient family that has been subjected to a large expansion in a restricted phylogenic group.

Linkage disequilibrium between minisatellite loci supports clonal evolution of Mycobacterium tuberculosis in a high tuberculosis incidence area.

Deciphering the structure of pathogen populations is instrumental for the understanding of the epidemiology and history of infectious diseases and for their control. Although Mycobacterium tuberculosis is the most widespread infectious agent in humans, its actual population structure has remained hypothetical until now because: (i) its structural genes are poorly polymorphic; (ii) adequate samples and appropriate statistics for population genetic analysis have not been considered. To investigate this structure, we analysed the statistical associations (linkage disequilibrium) between 12 independent M. tuberculosis minisatellite-like loci by high-throughput genotyping within a model population of 209 isolates representative of the genetic diversity in an area with a very high incidence of tuberculosis. These loci contain variable number tandem repeats (VNTRs) of genetic elements named mycobacterial interspersed repetitive units (MIRUs). Highly significant linkage disequilibrium was detected among the MIRU-VNTR loci in this model. This linkage disequilibrium was also evident when the MIRU-VNTR types were compared with the IS6110 restriction fragment length polymorphism types. These results support a predominant clonal evolution of M. tuberculosis.

Mycolic acid biosynthesis and enzymic characterization of the beta-ketoacyl-ACP synthase A-condensing enzyme from Mycobacterium tuberculosis.

Mycolic acids consist of long-chain alpha-alkyl-beta-hydroxy fatty acids that are produced by successive rounds of elongation catalysed by a type II fatty acid synthase (FAS-II). A key feature in the elongation process is the condensation of a two-carbon unit from malonyl-acyl-carrier protein (ACP) to a growing acyl-ACP chain catalysed by a beta-ketoacyl-ACP synthase (Kas). In the present study, we provide evidence that kasA from Mycobacterium tuberculosis encodes an enzyme that elongates in vivo the meromycolate chain, in both Mycobacterium smegmatis and Mycobacterium chelonae. We demonstrate that KasA belongs to the FAS-II system, which utilizes primarily palmitoyl-ACP rather than short-chain acyl-ACP primers. Furthermore, in an in vitro condensing assay using purified recombinant KasA, palmitoyl-AcpM and malonyl-AcpM, KasA was found to express Kas activity. Also, mutated KasA proteins, with mutation of Cys(171), His(311), Lys(340) and His(345) to Ala abrogated the condensation activity of KasA in vitro completely. Finally, purified KasA was highly sensitive to cerulenin, a well-known inhibitor of Kas, which may lead to the development of novel anti-mycobacterial drugs targeting KasA.