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Signalling by the Wnt family of secreted lipoproteins has essential functions in development and disease 1 . The canonical Wnt/β-catenin pathway requires a single-span transmembrane receptor, low-density lipoprotein (LDL)-receptor-related protein 6 (LRP6) 2 , 3 , 4 , whose phosphorylation at multiple PPPSP motifs is induced upon stimulation by Wnt and is critical for signal transduction 5 . The kinase responsible for LRP6 phosphorylation has not been identified. Here we provide biochemical and genetic evidence for a ‘dual-kinase’ mechanism for LRP6 phosphorylation and activation. Glycogen synthase kinase 3 (GSK3), which is known for its inhibitory role in Wnt signalling through the promotion of β-catenin phosphorylation and degradation, mediates the phosphorylation and activation of LRP6. We show that Wnt induces sequential phosphorylation of LRP6 by GSK3 and casein kinase 1, and this dual phosphorylation promotes the engagement of LRP6 with the scaffolding protein Axin. We show further that a membrane-associated form of GSK3, in contrast with cytosolic GSK3, stimulates Wnt signalling and Xenopus axis duplication. Our results identify two key kinases mediating Wnt co-receptor activation, reveal an unexpected and intricate logic of Wnt/β-catenin signalling, and illustrate GSK3 as a genuine switch that dictates both on and off states of a pivotal regulatory pathway.

The Wnt family of secreted signalling molecules are essential in embryo development and tumour formation 1 . The Frizzled (Fz) family of serpentine receptors function as Wnt receptors 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , but how Fz proteins transduce signalling is not understood. In Drosophila , arrow phenocopies the wingless (DWnt-1) phenotype 11 , and encodes a transmembrane protein 11 that is homologous to two members of the mammalian low-density lipoprotein receptor (LDLR)-related protein (LRP) family, LRP5 and LRP6 (refs 12 , 13 , 14 , 15 ). Here we report that LRP6 functions as a co-receptor for Wnt signal transduction. In Xenopus embryos, LRP6 activated Wnt–Fz signalling, and induced Wnt responsive genes, dorsal axis duplication and neural crest formation. An LRP6 mutant lacking the carboxyl intracellular domain blocked signalling by Wnt or Wnt–Fz, but not by Dishevelled or β-catenin, and inhibited neural crest development. The extracellular domain of LRP6 bound Wnt-1 and associated with Fz in a Wnt-dependent manner. Our results indicate that LRP6 may be a component of the Wnt receptor complex.

In humans, loss-of-function mutations in the gene encoding Wnt1 inducible signaling pathway protein 3 (WISP3) cause the autosomal-recessive skeletal disorder progressive pseudorheumatoid dysplasia (PPD). However, in mice there is no apparent phenotype caused by Wisp3 deficiency or overexpression. Consequently, the in vivo activities of Wisp3 have remained elusive. We cloned the zebrafish ortholog of Wisp3 and investigated its biologic activity in vivo using gain-of-function and loss-of-function approaches. Overexpression of zebrafish Wisp3 protein inhibited bone morphogenetic protein (BMP) and Wnt signaling in developing zebrafish. Conditioned medium-containing zebrafish and human Wisp3 also inhibited BMP and Wnt signaling in mammalian cells by binding to BMP ligand and to the Wnt coreceptors low-density lipoprotein receptor-related protein 6 (LRP6) and Frizzled, respectively. Wisp3 proteins containing disease-causing amino acid substitutions found in patients with PPD had reduced activity in these assays. Morpholino-mediated inhibition of zebrafish Wisp3 protein expression in developing zebrafish affected pharyngeal cartilage size and shape. These data provide a biologic assay for Wisp3, reveal a role for Wisp3 during zebrafish cartilage development, and suggest that dysregulation of BMP and/or Wnt signaling contributes to cartilage failure in humans with PPD.

To mitigate the impact of dementia, initiating early intervention is important. This study aims to investigate the associations between deterioration in oral function and cognitive decline in older outpatients whose oral health was maintained in the dental clinic. This study included 50 outpatients aged ≥65 years. We used the Japanese version of the Montreal Cognitive Assessment (MoCA-J) to assess cognitive decline. Oral function was evaluated by tongue pressure, masticatory performance, and swallowing ability. A full-mouth periodontal examination was conducted, and the occlusal support and number of teeth were recorded. Odds ratios (ORs) and 95% confidence intervals (CIs) for cognitive decline (MoCA-J score ≤25 points) were calculated using logistic regression models. The age, number of teeth, tongue pressure, and masticatory performance were significantly correlated with cognitive decline (p < 0.05). Logistic regression analyses revealed that cognitive decline was independently associated with age (OR: 1.25; 95% CI: 1.03–1.52; p = 0.024), number of teeth (OR = 0.83; 95% CI: 0.76–1.00; p = 0.047), and lower tongue pressure (OR: 0.87; 95% CI: 0.77–0.98; p = 0.022). Lower tongue pressure and a small number of remaining teeth may be associated with cognitive decline in Japanese outpatients.

Signalling by the Wnt family of secreted lipoproteins plays essential roles in development and disease1. The canonical Wnt/β-catenin pathway requires a single-span transmembrane receptor, LDL receptor related protein 6 (LRP6)2-4, whose phosphorylation at multiple PPPSP motifs is induced upon Wnt stimulation and critical for signal transduction5. The kinase responsible for LRP6 phosphorylation has not been identified. Here we provide biochemical and genetic evidence for a ‘dual-kinase’ mechanism for LRP6 phosphorylation and activation. Surprisingly, glycogen synthase kinase 3 (GSK3), which is known for its inhibitory role in Wnt signalling via promoting β-catenin phosphorylation and degradation, mediates LRP6 phosphorylation and activation. We demonstrate that Wnt induces sequential phosphorylation of LRP6 by GSK3 and casein kinase 1 (CK1), and this dual-phosphorylation promotes the engagement of LRP6 with the scaffolding protein Axin. We further show that a membrane-associated form of GSK3, contrary to cytosolic GSK3, stimulates Wnt signalling and Xenopus axis duplication. Our results identify two key kinases mediating Wnt coreceptor activation, reveal an unexpected and intricate logic of Wnt/β-catenin signalling, and illustrate GSK3 as a bona fide switch dictating both on and off states of a pivotal regulatory pathway.

In childhood acute lymphoblastic leukemia (ALL), TP53 gene mutation is associated with chemoresistance in a certain population of relapsed cases. To directly verify the association of TP53 gene mutation with chemoresistance of relapsed childhood ALL cases and improve their prognosis, the development of appropriate human leukemia models having TP53 mutation in the intrinsic gene is required. Here, we sought to introduce R248Q hotspot mutation into the intrinsic TP53 gene in an ALL cell line, 697, by applying a prime editing (PE) system, which is a versatile genome editing technology. The PE2 system uses an artificial fusion of nickase Cas9 and reverse‐transcriptase to directly place new genetic information into a target site through a reverse transcriptase template in the prime editing guide RNA (pegRNA). Moreover, in the advanced PE3b system, single guide RNA (sgRNA) matching the edited sequence is also introduced to improve editing efficiency. The initially obtained MDM2 inhibitor‐resistant PE3b‐transfected subline revealed disrupted p53 transactivation activity, reduced p53 target gene expression, and acquired resistance to chemotherapeutic agents and irradiation. Although the majority of the subline acquired the designed R248Q and adjacent silent mutations, the insertion of the palindromic sequence in the scaffold hairpin structure of pegRNA and the overlap of the original genomic DNA sequence were frequently observed. Targeted next‐generation sequencing reconfirmed frequent edit errors in both PE2 and PE3b‐transfected 697 cells, and it revealed frequent successful edits in HEK293T cells. These observations suggest a requirement for further modification of the PE2 and PE3b systems for accurate editing in leukemic cells. The TP53 gene is the most frequently mutated gene in many cancers, and R248Q hotspot mutation of the TP53 gene is reportedly developed in leukemia cells during chemotherapy in a certain population of childhood acute lymphoblastic leukemia (ALL) cases and subsequently results in disease relapse. To directly verify the association of R248Q hotspot mutation of the TP53 gene with chemoresistance in relapsed childhood ALL, we introduced it into the intrinsic TP53 gene of human ALL cell line by applying the prime editing (PE) system, an ideal and versatile genome editing technology, and obtained a subline in which transactivation activity and target gene expression of p53 protein were disrupted and, subsequently, sensitivities to chemotherapeutic agents and irradiation were significantly reduced. Of note, although the acquisition of the designed R248Q mutation as well as the intended additional silent mutation was confirmed as a result of PE in the obtained subline, we additionally noticed previously uncommon editing errors consisting of prime editing guide RNA sequence (part of the scaffold including the 3′ small hairpin structure) and overlapped original genomic DNA sequence, suggesting a requirement of further improvement of the PE system for applying it in cancer research.

Our study highlights the discovery of recurrent copy number alterations in noncoding regions, specifically blood enhancer cluster (BENC‐CNA), in B‐precursor acute lymphoblastic leukemia (BCP‐ALL) cell lines. We demonstrate that BENC‐CNA acts as a super‐enhancer, driving MYC expression and possibly contributing to the immortalization and proliferative advantage of BCP‐ALL cells in vitro.

•The development of 5-combined vaccine and others is of high priority in Japan.•The first vaccination of DPT-IPV should be given concomitantly with the Hib vaccine.•Gobik is the first DPT-IPV-Hib pentavalent vaccine approved in Japan.•Gobik simultaneously provide primary and booster immunizations as a single agent. This study investigated the immunogenicity and safety of a pentavalent vaccine Gobik (DPT-IPV-Haemophilus influenzae type b [Hib]) in healthy Japanese infants aged ≥ 2 and < 43 months using a concomitant vaccination with ActHIB® (Hib) and Tetrabik (DPT-IPV) as a comparator. This study was conducted as a phase 3, multicenter, active controlled, assessor-blinded, randomized, parallel-group study. Participants received a total of 4 subcutaneous doses (3 primary immunization doses and a booster dose) of either the experimental drug (DPT-IPV-Hib) or the active comparator (Hib + DPT-IPV). The primary endpoints were the anti-PRP antibody prevalence rate with ≥ 1 μg/mL, and the antibody prevalence rates against pertussis, diphtheria toxin, tetanus toxin, and attenuated poliovirus after the primary immunization. In 267 randomized participants (133 in the DPT-IPV-Hib group and 134 in the Hib + DPT-IPV group), the antibody prevalence rates after the primary immunization in both groups were 100.0 % and 88.7 % for anti-PRP antibody with ≥ 1 μg/mL, 99.2 % and 98.5 % against diphtheria toxin, and 100.0 % and 99.2 % against tetanus toxin, respectively. The antibody prevalence rates against pertussis and attenuated poliovirus were 100.0 % in both groups. The non-inferiority of the DPT-IPV-Hib group to the Hib + DPT-IPV group was verified for all measured antibodies. In both groups, all the GMTs of antibodies after the primary immunization were higher than those before the first dose, and those after the booster dose were higher than those after the primary immunization. No safety issues were identified. A single-agent Gobik, the first DPT-IPV-Hib pentavalent vaccine approved in Japan, was confirmed to simultaneously provide primary and booster immunizations against Hib infection, pertussis, diphtheria, tetanus, and poliomyelitis and to have a preventive effect and safety comparable to concomitant vaccination with Hib (ActHIB®) and DPT-IPV quadrivalent vaccine (Tetrabik).

Many breast cancer patients suffer from chemotherapy-induced hair loss. Accurate information about temporal changes in chemotherapy-induced hair loss is important for supporting patients scheduled to receive chemotherapy, because it helps them to prepare. However, accurate information, on issues such as the frequency of hair loss after chemotherapy, when regrowth starts, the condition of regrown hair, and the frequency of incomplete hair regrowth, is lacking. This study aimed to clarify the long-term temporal changes in chemotherapy-induced hair loss using patient-reported outcomes for chemotherapy-induced hair loss. We conducted a multicenter, cross-sectional questionnaire survey. Disease-free patients who had completed adjuvant chemotherapy consisting of anthracycline and/or taxanes for breast cancer within the prior 5 years were enrolled from 47 hospitals and clinics in Japan. Descriptive statistics were obtained in this study. The study is reported according to the STROBE criteria. The response rate was 81.5% (1511/1853), yielding 1478 questionnaires. Hair loss occurred in 99.9% of patients. The mean time from chemotherapy until hair loss was 18.0 days. Regrowth of scalp hair occurred in 98% of patients. The mean time from the completion of chemotherapy to the beginning of regrowth was 3.3 months. Two years after chemotherapy completion, the scalp-hair recovery rate was <30% in approximately 4% of patients, and this rate showed no improvement 5 years after chemotherapy. Eighty-four percent of the patients initially used wigs, decreasing to 47% by 1 year after chemotherapy and 15.2% after 2 years. The mean period of wig use was 12.5 months. However, a few patients were still using wigs 5 years after completing chemotherapy. Our survey focused on chemotherapy-induced hair loss in breast cancer patients. We believe these results to be useful for patients scheduled to receive chemotherapy.

Non-occupational exposure to cadmium has been suspected to be a risk factor for breast cancer. The present study examined the association between urinary cadmium level and the risk of breast cancer in a case–control study among Japanese women. Cases were 153 women newly diagnosed and histologically confirmed with breast cancer at a general hospital in Gifu, Japan. A total of 431 controls individually matched to cases by age, menopausal status, and the period of urine sampling were selected from those who attended a breast cancer mass screening at this hospital. Urinary cadmium levels were measured using spot urine samples. Spot urine samples were collected from cases after surgery but before any cancer therapy. For controls, spot urine samples were obtained at the date of the screening visit. Information on known or suggested breast cancer risk factors was obtained by a self-administered questionnaire. The odds ratios (ORs) and 95 % confidence intervals (CIs) of breast cancer according to the tertile of the creatinine-adjusted cadmium level were calculated using conditional logistic regression models. Women in the highest tertile of the creatinine-adjusted cadmium level (>2.620 μg/g) had significantly elevated OR of breast cancer relative to those in the lowest tertile (<1.674 μg/g) after controlling for covariates [OR = 6.05, (95 % CI 2.90, 12.62)]. The trend of increase in risk with increasing cadmium level was also statistically significant [OR = 1.67, (95 % CI 1.39, 2.01) for every 1.0 μg/g increase in urinary cadmium level, P -trend <0.01]. These data suggested that exposure to cadmium was associated with a risk of breast cancer in Japanese women.