TRIM37 interacts with EZH2 to epigenetically suppress PTCH1 and regulate stemness in glioma stem cells through sonic hedgehog pathway.

BACKGROUND: Glioma stem cells (GSCs), which are known for their therapy resistance, play a substantial role in treatment inefficacy for glioblastoma multiforme (GBM). TRIM37, a member of the tripartite motif (TRIM) protein family initially linked to a rare growth disorder, has been recognized for its oncogenic role. However, the mechanism by which TRIM37 regulates tumor growth in glioma and GSCs is unclear. METHODS: For the in vitro experiments, gene expression was measured by western blotting, RT-qPCR, and immunofluorescence. Cell viability was detected by CCK-8, and cell apoptosis was detected by flow cytometry. The interaction between Enhancer of Zeste Homolog 2 (EZH2) and TRIM37 was verified by co-immunoprecipitation (Co-IP). The interaction between EZH2 and the PTCH1 promoter was verified using dual-luciferase reporter assay and chromatin immunoprecipitation (ChIP). For the in vivo experiments, an orthotopically implanted glioma mouse model was used to validate tumor growth. RESULTS: The expression of TRIM37 is higher in GSCs compared with matched non-GSCs. TRIM37 knockdown promotes apoptosis, decreased stemness in GSCs, and reduces tumor growth in GSCs xenografts of nude mice. TRIM37 and EZH2 co-localize in the nucleus and interact with each other. TRIM37 knockdown or EZH2 inhibition downregulates the protein expressions associated with the Sonic Hedgehog (SHH) pathway. EZH2 epigenetically downregulates PTCH1 to activate SHH pathway in GSCs. CONCLUSIONS: TRIM37 maintains the cell growth and stemness in GSCs through the interaction with EZH2. EZH2 activates SHH stem cell signaling pathway by downregulating the expression of SHH pathway suppressor PTCH1. Our findings suggest that TRIM37 may be a potential therapeutic target for GBM.

PTCH/SMO gene mutations in odontogenic keratocysts and drug interventions.

BACKGROUND: Odontogenic keratocysts (OKCs) are odontogenic jaw lesions that cause destruction and dysfunction of the jawbone. OKCs can be sporadic or associated with nevoid basic cell carcinoma syndrome (NBCCS). However, the factors that initiate OKCs and the mechanism of cyst formation remain unclear. Here, we investigated the impact of PTCH1 and SMO mutations on disease progression, as well as the effects of sonic hedgehog (SHH) signaling pathway inhibitors GDC-0449 and GANT61 on OKC fibroblasts. METHODS: Eight sporadic OKC fibroblasts without gene mutations were used as the control, and six NBCCS-related fibroblasts were cultured in vitro. The effect of PTCH1 non-truncated mutation 3499G>A (p.G1167R) and SMO c.2081C>G (p.P694R) mutation on OKC fibroblast proliferation was examined by EdU assay. CCK8 and wound-healing assays detected the effects of OKC fibroblasts carrying PTCH1 c.3499G>A (p.G1167R) and SMO c.2081C>G (p.P694R) mutations on the proliferation and migration of HaCaT cells after co-culture. Quantitative real-time PCR detected the effects of GDC-0449 or GANT61 on the SHH signaling pathway in NBCCS-related OKCs with PTCH1 truncated mutations and PTCH1 c.3499G>A (p.G1167R) and/or SMO c.2081C>G (p.P694R) mutations. RESULTS: PTCH1 c.3499G>A (p.G1167R) and SMO c.2081C>G (p.P694R) promoted the proliferation of OKC fibroblasts. The proliferation and migration of HaCaT cells were affected by NBCCS-related OKC fibroblasts carrying PTCH1 c.3499G>A (p.G1167R) and SMO c.2081C>G (p.P694R) mutations. GDC-0449 significantly inhibited the SHH signaling pathway in NBCCS-related OKC fibroblasts with PTCH1 truncated mutations. An NBCCS-related OKC carrying PTCH1 c.3499G>A (p.G1167R) and SMO c.2081C>G (p.P694R) mutations were resistant to GDC-0449 but inhibited by GANT61. CONCLUSIONS: Genetic mutations in OKC fibroblasts may affect the biological behavior of epithelial and stromal cells and cause disease. GDC-0449 could be used to treat OKCs, especially NBCCS-related OKCs with PTCH1 truncated mutations. SMO c.2081C>G (p.P694R) may lead to resistance to GDC-0449; however, GANT61 may be used as an alternative inhibitor.

Gene-Specific DNA Methylation Profiles in Pediatric Medulloblastomas.

INTRODUCTION: Medulloblastoma is the most common pediatric central nervous tumor of high malignancy that has been classified into both histological subtypes and molecular subgroups by the 2016 World Health Organization classification. However, there is a still need to understand the genomic characteristics and predict the clinical course. The aim of the study is to investigate the significance of the methylation profiles in molecular subclassification and precision medicine of the disease. METHODS: The study enrolled 47 pediatric medulloblastoma patients. DNA methylation levels of KLF4, SPINT2, RASSF1A, EZH2, ZIC2, and PTCH1 genes were analyzed using methylation-specific pyrosequencing. The significance of the statistical relationship between methylation profiles and clinicopathological parameters including molecular subgroups and histological subtypes, the status of metastasis, and event-free survival were analyzed. RESULTS: DNA methylation analysis demonstrated that KLF4, PTCH1, and ZIC2 hypermethylation were associated with the SHH-activated subgroup, whereas both SPINT2 and RASSF1A hypermethylation were associated with metastatic disease. EZH2 gene was not methylated in any of the samples. CONCLUSION: We think that customized DNA methylation profiling may be a useful tool in the molecular subclassification of pediatric medulloblastoma and a potential technical approach in precision medicine.

Protective role of SIRT1-mediated Sonic Hedgehog signaling pathway in the preeclampsia rat models.

OBJECTIVE: To explore the role of silent information regulator 1 (SIRT1)-mediated Sonic Hedgehog (SHH) pathway in reduced uterine perfusion pressure (RUPP) model of preeclampsia (PE) in rats. METHODS: The pregnant rats were divided into sham, RUPP, RUPP + rSIRT1 (recombinant SIRT1 protein), RUPP + rSHH (recombinant SHH protein), and RUPP + rSIRT1+ Cy (cyclopamine, an SHH pathway inhibitor) groups, followed by the determination of mean arterial pressure (MAP) and pregnancy outcome. The gene or protein expression was determined by enzyme-linked immunosorbent assay (ELISA), quantitative reverse transcription-polymerase chain reaction (qRT-PCR), or Western blotting. RESULTS: RUPP rats showed increases MAP with the lower levels of vascular endothelial growth factor (VEGF) and nitrite and nitrate (NOx), as well as the higher levels of soluble FMS-like tyrosine kinase-1 (sFlt-1), tumor necrosis factor (TNF)-α, and interleukin (IL)-6 in maternal plasma, which was attenuated after rSIRT1 or rSHH treatment. Besides, the improvement in the pregnancy outcome was seen in the rats from the RUPP + rSIRT1/rSHH groups as compared with the RUPP group. However, the therapeutic effect of rSIRT1 was reversed by cyclopamine. Placenta tissues of RUPP rats manifested the down-regulations of SIRT1, Patched-1 (PTCH1), and GLI family zinc finger 2 (GLI2), which were up-regulated in the RUPP + rSIRT1 group. CONCLUSION: SIRT1 was down-regulated while SHH pathway was inhibited in the placental tissue of PE rats. SIRT1 improved the blood pressure, angiogenic imbalance, inflammation, and pregnancy outcome in PE rats via SHH pathway, supporting its potential use for the treatment of PE.

Molecular Bases of Human Malformation Syndromes Involving the SHH Pathway: GLIA/R Balance and Cardinal Phenotypes.

Human hereditary malformation syndromes are caused by mutations in the genes of the signal transduction molecules involved in fetal development. Among them, the Sonic hedgehog (SHH) signaling pathway is the most important, and many syndromes result from its disruption. In this review, we summarize the molecular mechanisms and role in embryonic morphogenesis of the SHH pathway, then classify the phenotype of each malformation syndrome associated with mutations of major molecules in the pathway. The output of the SHH pathway is shown as GLI activity, which is generated by SHH in a concentration-dependent manner, i.e., the sum of activating form of GLI (GLIA) and repressive form of GLI (GLIR). Which gene is mutated and whether the mutation is loss-of-function or gain-of-function determine in which concentration range of SHH the imbalance occurs. In human malformation syndromes, too much or too little GLI activity produces symmetric phenotypes affecting brain size, craniofacial (midface) dysmorphism, and orientation of polydactyly with respect to the axis of the limb. The symptoms of each syndrome can be explained by the GLIA/R balance model.

Extracephalic manifestations of nonchromosomal, nonsyndromic holoprosencephaly.

Nonchromosomal, nonsyndromic holoprosencephaly (NCNS-HPE) has traditionally been considered as a condition of brain and craniofacial maldevelopment. In this review, we present the results of a comprehensive literature search supporting a wide spectrum of extracephalic manifestations identified in patients with NCNS-HPE. These manifestations have been described in case reports and in large cohorts of patients with "single-gene" mutations, suggesting that the NCNS-HPE phenotype can be more complex than traditionally thought. Likely, a complex network of interacting genetic variants and environmental factors is responsible for these systemic abnormalities that deviate from the usual brain and craniofacial findings in NCNS-HPE. In addition to the systemic consequences of pituitary dysfunction (as a direct result of brain midline defects), here we describe a number of extracephalic findings of NCNS-HPE affecting various organ systems. It is our goal to provide a guide of extracephalic features for clinicians given the important clinical implications of these manifestations for the management and care of patients with HPE and their mutation-positive relatives. The health risks associated with some manifestations (e.g., fatty liver disease) may have historically been neglected in affected families.

Prognostic significance of the methylation of Wnt pathway antagonists-CXXC4, DACT2, and the inhibitors of sonic hedgehog signaling-ZIC1, ZIC4, and HHIP in head and neck squamous cell carcinomas.

OBJECTIVES: Aberrations in Wnt and Shh signaling pathways are related to the pathogenesis of head and neck carcinomas, and their activation frequently results from epigenetic alterations. This study aimed to assess the frequency of methylation of negative regulators of Wnt signaling: CXXC4, DACT2, HDPR1, and FBXW11 and Shh signaling: HHIP, PTCH1, SUFU, ZIC1, and ZIC4 and correlate it with clinicopathological features in this group of patients. MATERIALS AND METHODS: Methylation-specific PCR was used to detect gene promoter methylation, and real-time PCR was used to assess gene expression level. RESULTS: The analysis of the occurrence of gene promoter methylation in head and neck carcinoma cell lines indicated that CXXC4, DACT2, HHIP, ZIC1, and ZIC4 are methylated in these tumors. These genes were further analyzed in tumor sections from oral and laryngeal cancer patients. Gene methylation rate was higher in laryngeal tumors. The methylation index in tumor samples correlated with the overall survival in a subgroup of oral cancer patients who died of the disease. Moreover, ZIC4 methylation correlated with lymph node involvement in oral cancer patients. CONCLUSIONS: Our findings corroborate that the activation of Wnt signaling in head and neck squamous cell carcinoma (HNSCC) is related to epigenetic silencing of its negative regulators. Moreover, the results indicate that the same mechanism of activation may operate in the case of Shh signaling. CLINICAL RELEVANCE: The methylation of ZIC4 may be considered a new prognostic marker in oral cavity and oropharyngeal tumors. Further investigations should determine the diagnostic significance of methylation of ZIC4, HHIP, and DACT2 in head and neck carcinomas.

Scutellaria barbata D. Don inhibits tumor angiogenesis via suppression of Hedgehog pathway in a mouse model of colorectal cancer.

Angiogenesis, which plays a critical role during tumor development, is tightly regulated by the Sonic Hedgehog (SHH) pathway, which has been known to malfunction in many types of cancer. Therefore, inhibition of angiogenesis via modulation of the SHH signaling pathway has become very attractive for cancer chemotherapy. Scutellaria barbata D. Don (SB) has long been used in China to treat various cancers including colorectal cancer (CRC). Our published data suggested that the ethanol extract of SB (EESB) is able to induce apoptosis of colon cancer cells and inhibit angiogenesis in a chick embryo chorioallantoic membrane model. To further elucidate the precise mechanisms of its anti-tumor activity, in the present study we used a CRC mouse xenograft model to evaluate the effect of EESB on tumor growth and angiogenesis in vivo. Our current data indicated that EESB reduces tumor size without affecting on the body weight gain in CRC mice. In addition, EESB treatment suppresses the expression of key mediators of the SHH pathway in tumor tissues, which in turn resulted in the inhibition of tumor angiogenesis. Furthermore, EESB treatment inhibits the expression of vascular endothelial growth factor A (VEGF-A), an important target gene of SHH signaling and functioning as one of the strongest stimulators of angiogenesis. Our findings suggest that inhibition of tumor angiogenesis via suppression of the SHH pathway might be one of the mechanisms by which Scutellaria barbata D. Don can be effective in the treatment of cancers.

ISL1 Promotes Human Glioblastoma-Derived Stem Cells' Self-Renewal by Activation of Sonic Hedgehog/GLI1 Function.

Glioblastoma (GBM), the most aggressive primary heterogeneous primary brain tumor, is a glioma subtype that originates from the glial cells of the central nervous system. Glioblastoma stem cells (GSCs), situated at the top of the hierarchy, initiate and maintain the tumor and are largely accountable for GBM resistance to the mainstay treatment and recurrence. The LIM homeobox transcription factor islet 1 (ISL1) induces tumorigenicity in various tumors; however, its function in GSCs has been less reported. We aimed to generate GSCs from surgical specimens of human GBM and investigate the effect of ISL1 knockdown on GSCs. We established patient-derived GSCs, determined cancer stem cell marker expression, and immunostained GSCs to assess cell viability and apoptosis. We demonstrated that ISL1 deletion decreased the GSC viability and proliferation, and upregulated apoptosis. Moreover, we performed enzyme-linked immunosorbent assay and western blotting and found that ISL1 knockdown affected the expression of sonic hedgehog (SHH) and its downstream regulator GLI1, and further validated these results by supplementing the cells with recombinant SHH. Our results suggested that ISL1 played a critical role in regulating GBM growth and that an ISL1/SHH/GLI1 pathway was required for the maintenance of GBM progression and malignancy. The regulation of GSC growth through ISL1 might be a mechanism of interest for future therapeutic studies.

Potassium Channel KCNH1 Activating Variants Cause Altered Functional and Morphological Ciliogenesis.

The primary cilium is a non-motile sensory organelle that extends from the surface of most vertebrate cells and transduces signals regulating proliferation, differentiation, and migration. Primary cilia dysfunctions have been observed in cancer and in a group of heterogeneous disorders called ciliopathies, characterized by renal and liver cysts, skeleton and limb abnormalities, retinal degeneration, intellectual disability, ataxia, and heart disease and, recently, in autism spectrum disorder, schizophrenia, and epilepsy. The potassium voltage-gated channel subfamily H member 1 (KCNH1) gene encodes a member of the EAG (ether-à-go-go) family, which controls potassium flux regulating resting membrane potential in both excitable and non-excitable cells and is involved in intracellular signaling, cell proliferation, and tumorigenesis. KCNH1 missense variants have been associated with syndromic neurodevelopmental disorders, including Zimmermann-Laband syndrome 1 (ZLS1, MIM #135500), Temple-Baraitser syndrome (TMBTS, MIM #611816), and, recently, with milder phenotypes as epilepsy. In this work, we provide evidence that KCNH1 localizes at the base of the cilium in pre-ciliary vesicles and ciliary pocket of human dermal fibroblasts and retinal pigment epithelial (hTERT RPE1) cells and that the pathogenic missense variants (L352V and R330Q; NP_002229.1) perturb cilia morphology, assembly/disassembly, and Sonic Hedgehog signaling, disclosing a multifaceted role of the protein. The study of KCNH1 localization, its functions related to primary cilia, and the alterations introduced by mutations in ciliogenesis, cell cycle coordination, cilium morphology, and cilia signaling pathways could help elucidate the molecular mechanisms underlying neurological phenotypes and neurodevelopmental disorders not considered as classical ciliopathies but for which a significant role of primary cilia is emerging.

Molecular Targeted Therapies: Time for a Paradigm Shift in Medulloblastoma Treatment?

Medulloblastoma is a rare malignancy of the posterior cranial fossa. Although until now considered a single disease, according to the current WHO classification, it is a heterogeneous tumor that comprises multiple molecularly defined subgroups, with distinct gene expression profiles, pathogenetic driver alterations, clinical behaviors and age at onset. Adult medulloblastoma, in particular, is considered a rarer "orphan" entity in neuro-oncology practice because while treatments have progressively evolved for the pediatric population, no practice-changing prospective, randomized clinical trials have been performed in adults. In this scenario, the toughest challenge is to transfer the advances in cancer genomics into new molecularly targeted therapeutics, to improve the prognosis of this neoplasm and the treatment-related toxicities. Herein, we focus on the recent advances in targeted therapy of medulloblastoma based on the new and deeper knowledge of disease biology.

Adult Medulloblastoma: Updates on Current Management and Future Perspectives.

Medulloblastoma (MB) is a malignant embryonal tumor of the posterior fossa belonging to the family of primitive neuro-ectodermic tumors (PNET). MB generally occurs in pediatric age, but in 14-30% of cases, it affects the adults, mostly below the age of 40, with an incidence of 0.6 per million per year, representing about 0.4-1% of tumors of the nervous system in adults. Unlike pediatric MB, robust prospective trials are scarce for the post-puberal population, due to the low incidence of MB in adolescent and young adults. Thus, current MB treatments for older patients are largely extrapolated from the pediatric experience, but the transferability and applicability of these paradigms to adults remain an open question. Adult MB is distinct from MB in children from a molecular and clinical perspective. Here, we review the management of adult MB, reporting the recent published literature focusing on the effectiveness of upfront chemotherapy, the development of targeted therapies, and the potential role of a reduced dose of radiotherapy in treating this disease.

MEVITEM-a phase I/II trial of vismodegib + temozolomide vs temozolomide in patients with recurrent/refractory medulloblastoma with Sonic Hedgehog pathway activation.

BACKGROUND: Vismodegib specifically inhibits Sonic Hedgehog (SHH). We report results of a phase I/II evaluating vismodegib + temozolomide (TMZ) in immunohistochemically defined SHH recurrent/refractory adult medulloblastoma. METHODS: TMZ-naïve patients were randomized 2:1 to receive vismodegib + TMZ (arm A) or TMZ (arm B). Patients previously treated with TMZ were enrolled in an exploratory cohort of vismodegib (arm C). If the safety run showed no excessive toxicity, a Simon's 2-stage phase II design was planned to explore the 6-month progression-free survival (PFS-6). Stage II was to proceed if arm A PFS-6 was ≥3/9 at the end of stage I. RESULTS: A total of 24 patients were included: arm A (10), arm B (5), and arm C (9). Safety analysis showed no excessive toxicity. At the end of stage I, the PFS-6 of arm A was 20% (2/10 patients, 95% unilateral lower confidence limit: 3.7%) and the study was prematurely terminated. The overall response rates (ORR) were 40% (95% CI, 12.2-73.8) and 20% (95% CI, 0.5-71.6) in arm A and B, respectively. In arm C, PFS-6 was 37.5% (95% CI, 8.8-75.5) and ORR was 22.2% (95% CI, 2.8-60.0). Among 11 patients with an expected sensitivity according to new generation sequencing (NGS), 3 had partial response (PR), 4 remained stable disease (SD) while out of 7 potentially resistant patients, 1 had PR and 1 SD. CONCLUSION: The addition of vismodegib to TMZ did not add toxicity but failed to improve PFS-6 in SHH recurrent/refractory medulloblastoma. Prediction of sensitivity to vismodegib needs further refinements.

Targeted Inhibition of Fibroblast Growth Factor Receptor 1-GLI Through AZD4547 and GANT61 Modulates Breast Cancer Progression.

The underlying mechanism of fibroblast growth factor receptor 1 (FGFR1) mediated carcinogenesis is still not fully understood. For instance, FGFR1 upregulation leads to endocrine therapy resistance in breast cancer patients. The current study aimed to identify FGFR1-linked genes to devise improved therapeutic strategies. RNA-seq and microarray expression data of 1,425 breast cancer patients from two independent cohorts were downloaded for the analysis. Gene Set Enrichment Analysis (GSEA) was performed to identify differentially expressed pathways associated with FGFR1 expression. Validation was done using 150 fresh tumor biopsy samples of breast cancer patients. The clinical relevance of mRNA and protein expression of FGFR1 and its associated genes were also evaluated in mouse embryonic fibroblasts (MEFs) and breast cancer cell line (MDA-MB-231). Furthermore, MDA-MB-231 cell line was treated with AZD4547 and GANT61 to identify the probable role of FGFR1 and its associated genes on cells motility and invasion. According to GSEA results, SHH pathway genes were significantly upregulated in FGFR1 patients in both discovery cohorts of breast cancer. Statistical analyses using both discovery cohorts and 150 fresh biopsy samples revealed strong association of FGFR1 and GLI1, a member of SHH pathway. The increase in the expression of these molecules was associated with poor prognosis, lymph node involvement, late stage, and metastasis. Combined exposures to AZD4547 (FGFR1 inhibitor) and GANT61 (GLI1 inhibitor) significantly reduced cell proliferation, cell motility, and invasion, suggesting molecular crosstalk in breast cancer progression and metastasis. A strong positive feedback mechanism between FGFR1-GLI1 axis was observed, which significantly increased cell proliferation and metastasis. Targeting FGFR1-GLI1 simultaneously will significantly improve the prognosis of breast cancer in patients.

Anti-hepatoma effect of taccalonolide A through suppression of sonic hedgehog pathway.

Taccalonolide A has been reported to have anti-tumour efficiency. However, the underlying mechanism for taccalonolides A therapy of hepatocellular carcinoma (HCC) is still obscure. Cell viability was evaluated by cell counting kit-8 (CCK-8) assay. Apoptosis was determined by flow cytometry. Protein expression of B cell lymphoma (Bcl-2), Bcl-2 associated X (Bax), sonic hedgehog (Shh), Smoothened (Smo) and Gli family zinc finger 1 (Gli1) was analyzed by western blot. The expression of Shh, Smo and Gli1 mRNA was determined using quantitative real-time polymerase chain reaction (qRT-PCR). Results showed that taccalonolide A inhibited cell proliferation, induced apoptosis and cell cycle arrest at the G0/G1 phase, and improved the cytotoxicity of sorafenib in HCC cells. The expressions of Shh, Smo, Gli1 mRNA and protein were decreased after taccalonolide A treatment. More importantly, activation of the Shh pathway attenuated taccalonolide A-induced inhibition on cell viability and promotion on apoptosis and cell cycle arrest in HCC. Also, activation of the Shh pathway neutralized the effect of taccalonolide A on sorafenib cytotoxicity in HCC. We clarified that taccalonolide A suppressed cell viability facilitated apoptosis, and improved the cytotoxicity of sorafenib in HCC by inhibition of the activation of the Shh pathway, providing alternative treatments for HCC.

Dihydroartemisinin Sensitizes Esophageal Squamous Cell Carcinoma to Cisplatin by Inhibiting Sonic Hedgehog Signaling.

Platinum-based regimens have been routinely used in the clinical treatment of patients with esophageal squamous cell carcinoma (ESCC). However, administration of these drugs is frequently accompanied by drug resistance. Revealing the underlying mechanisms of the drug resistance and developing agents that enhance the sensitivity to platinum may provide new therapeutic strategies for the patients. In the present study, we found that the poor outcome of ESCC patients receiving platinum-based regimens was associated with co-expression of Shh and Sox2. The sensitivity of ESCC cell lines to cisplatin was related to their activity of Shh signaling. Manipulating of Shh expression markedly changed the sensitivity of ESCC cells to platinum. Continuous treatment with cisplatin resulted in the activation of Shh signaling and enhanced cancer stem cell-like phenotypes in ESCC cells. Dihydroartemisinin (DHA), a classic antimalarial drug, was identified as a novel inhibitor of Shh pathway. Treatment with DHA attenuated the cisplatin-induced activation of the Shh pathway in ESCC cells and synergized the inhibitory effect of cisplatin on proliferation, sphere and colony formation of ALDH-positive ESCC cells in vitro and growth of ESCC cell-derived xenograft tumors in vivo. Taken together, these results demonstrate that the Shh pathway is an important player in cisplatin-resistant ESCC and DHA acts as a promising therapeutic agent to sensitize ESCC to cisplatin treatment.

A Novel Cerebroprotein Hydrolysate, CH1, Ameliorates Chronic Focal Cerebral Ischemia Injury by Promoting White Matter Integrity via the Shh/Ptch-1/Gli-1 Signaling Pathway.

PURPOSE: Strokes are devastating as there are no current therapies to prevent long-term neurological deficits. Previous studies reported that cerebroprotein hydrolysate (CH) plays a role in neuronal protection in acute phase after ischemic stroke, while the long-term effects of CH upon brain plasticity and neurological outcomes after stroke are still uncertain. To address these gaps, we assessed the effect of a new cerebroprotein hydrolysate, CH1, on long-term gray and white matter integrity as well as axonal plasticity in the late phase after ischemic stroke and the potential mechanisms. METHODS: Adult male mice were subjected to permanent distal middle cerebral artery occlusion (dMCAO), followed by daily intraperitoneal injection of CH1 for 14 days. Motor function was measured weekly through behavioral neurological evaluations. Gray matter intensity and white matter intensity were examined by immunofluorescence staining. The sonic hedgehog (Shh) inhibitor cyclopamine (CYC) was injected to determine the involvement of the Shh pathway in the therapeutic effects of CH1. RESULTS: We found that intraperitoneal delivery of CH1, compared to vehicle administration, significantly improved long-term neurological outcomes at various times and promoted neuronal viability at 14 days but not at 28 days after stroke. Importantly, CH1 mitigated stroke-induced white matter injury and facilitated axonal plasticity in the late stage after stroke. CONCLUSION: These results unveil a previously unappreciated role for CH in the repair of white matter and brain plasticity after stroke.

Pik3ca mutations significantly enhance the growth of SHH medulloblastoma and lead to metastatic tumour growth in a novel mouse model.

Medulloblastoma (MB) is the most frequent malignant brain tumour in children with a poor outcome. Divided into four molecular subgroups, MB of the Sonic hedgehog (SHH) subgroup accounts for approximately 25% of the cases and is driven by mutations within components of the SHH pathway, such as its receptors PTCH1 or SMO. A fraction of these cases additionally harbour PIK3CA mutations, the relevance of which is so far unknown. To unravel the role of Pik3ca mutations alone or in combination with a constitutively activated SHH signalling pathway, transgenic mice were used. These mice show mutated variants within Smo, Ptch1 or Pik3ca genes in cerebellar granule neuron precursors, which represent the cellular origin of SHH MB. Our results show that Pik3ca mutations alone are insufficient to cause developmental alterations or to initiate MB. However, they significantly accelerate the growth of Shh MB, induce tumour spread throughout the cerebrospinal fluid, and result in lower survival rates of mice with a double Pik3caH1047R/SmoM2 or Pik3caH1047R/Ptch1 mutation. Therefore, PIK3CA mutations in SHH MB may represent a therapeutic target for first and second line combination treatments.

Cholest-4,6-Dien-3-One Promote Epithelial-To-Mesenchymal Transition (EMT) in Biliary Tree Stem/Progenitor Cell Cultures In Vitro.

Human biliary tree stem/progenitor cells (hBTSCs), reside in peribiliary glands, are mainly stimulated by primary sclerosing cholangitis (PSC) and cholangiocarcinoma. In these pathologies, hBTSCs displayed epithelial-to-mesenchymal transition (EMT), senescence characteristics, and impaired differentiation. Here, we investigated the effects of cholest-4,6-dien-3-one, an oxysterol involved in cholangiopathies, on hBTSCs biology. hBTSCs were isolated from donor organs, cultured in self-renewal control conditions, differentiated in mature cholangiocytes by specifically tailored medium, or exposed for 10 days to concentration of cholest-4,6-dien-3-one (0.14 mM). Viability, proliferation, senescence, EMT genes expression, telomerase activity, interleukin 6 (IL6) secretion, differentiation capacity, and HDAC6 gene expression were analyzed. Although the effect of cholest-4,6-dien-3-one was not detected on hBTSCs viability, we found a significant increase in cell proliferation, senescence, and IL6 secretion. Interestingly, cholest-4.6-dien-3-one impaired differentiation in mature cholangiocytes and, simultaneously, induced the EMT markers, significantly reduced the telomerase activity, and induced HDAC6 gene expression. Moreover, cholest-4,6-dien-3-one enhanced bone morphogenic protein 4 (Bmp-4) and sonic hedgehog (Shh) pathways in hBTSCs. The same pathways activated by human recombinant proteins induced the expression of EMT markers in hBTSCs. In conclusion, we demonstrated that chronic exposition of cholest-4,6-dien-3-one induced cell proliferation, EMT markers, and senescence in hBTSC, and also impaired the differentiation in mature cholangiocytes.

ISLET1-Dependent ß-Catenin/Hedgehog Signaling Is Required for Outgrowth of the Lower Jaw.

Mandibular patterning information initially resides in the epithelium during development. However, how transcriptional regulation of epithelium-derived signaling controls morphogenesis of the mandible remains elusive. Using ShhCre to target the mandibular epithelium, we ablated transcription factor Islet1, resulting in a distally truncated mandible via unbalanced cell apoptosis and decreased cell proliferation in the distal mesenchyme. Loss of Islet1 caused a lack of cartilage at the distal tip, leading the fusion of two growing mandibular elements surrounding the rostral process of Meckel's cartilage. Loss of Islet1 results in dysregulation of mesenchymal genes important for morphogenesis of the mandibular arch. We revealed that Islet1 is required for the activation of epithelial ß-catenin signaling via repression of Wnt antagonists. Reactivation of ß-catenin in the epithelium of the Islet1 mutant rescued mandibular morphogenesis through sonic hedgehog (SHH) signaling to the mesenchyme. Furthermore, overexpression of a transgenic hedgehog ligand in the epithelium also partially restored outgrowth of the mandible. These data reveal functional roles for an ISLET1-dependent network integrating ß-catenin/SHH signals in mesenchymal cell survival and outgrowth of the mandible during development.