Glioblastoma (GBM) is an aggressive human malignancy. Recent advances in GBM research have highlighted innovative therapeutic approaches, including the use of small molecules that eliminate GBM in mouse models. However, there are few reports on the restoration of lost neuronal functions in patients. Considering that GBM contains GBM-initiating cells (GICs) with characteristics of both cancer and neural stem cells, we investigated whether GICs could be redirected toward non-tumorigenic neurons to support the preservation of neural function in the brain with GBM. We demonstrated that the neuronal differentiation inducer Isoxazole 9 (ISX9) effectively induced GICs to differentiate into neurons, accompanied by significant changes in their gene expression profiles. The sequential application of ISX9 and the DHODH inhibitor brequinar (BRQ), which successfully eradicated undifferentiated GICs, not only promoted neuronal differentiation but also inhibited GIC tumorigenesis in the mouse brain, leading to prolonged survival and preservation of motor function in tumor-bearing mice. Furthermore, pathological analysis revealed that this combination not only reduced the size of GIC brain tumors but also facilitated the formation of synapse-like structural contacts between GIC-derived cells and host mouse neurons, suggesting remodeling of the tumor-neural interface within the tumor-developed area. Collectively, these findings suggest that the modulation of tumorigenic GIC differentiation may represent a strategy to preserve neural circuit integrity within the tumor-bearing brain.

Glioblastoma (GBM) is the most common malignant brain tumor in adults. Despite multimodal treatments, including surgery, radiation therapy, and temozolomide (TMZ) chemotherapy, the median survival remains poor at approximately 15 months. One reason for the therapeutic resistance is the existence of GBM-initiating cells (GICs) within the tumor. Therefore, understanding the molecular insights of how GICs contribute to the therapy recurrence is crucial for developing new therapeutic strategies. Comparing expression profiles of TMZ-resistant GICs (GICRs) with those of GICs, we identified membrane-associated protein 17 (MAP17) as a new factor that is exclusively expressed in GICRs. We show that overexpression of MAP17 in GICs significantly increased their proliferation, TMZ resistance, and tumorigenicity, whereas its knockdown impaired these properties, indicating that MAP17 plays a critical role in both TMZ resistance and tumorigenicity of GICs. We also show that MAP17 increased the expression of anti-apoptotic protein BCL2 through the activation of RELA-dependent NF-κB pathway in GICs. Furthermore, we demonstrate that overexpression of BCL2 increased TMZ resistance in GICs and their tumorigenicity, while its knockdown deprived these malignant characters in GICRs. Taken together, these findings identify a novel signaling pathway, MAP17-NF-κB-BCL2, that controls TMZ resistance and tumorigenicity of GICs.

Current treatments for Alzheimer's disease (AD) primarily focus on slowing disease progression, emphasizing the urgent need for a reliable diagnostic method for early-stage Alzheimer's (EAD). Our investigation, which is based on the finding that the secretory protein Esophageal Cancer Related Gene 4 (ECRG4) is elevated in the hippocampus of AD patients, led to the creation of a novel ELISA system for ECRG4 detection. We observed that ECRG4 peptides, particularly the fragment spanning amino acids 108-132, were elevated in the plasma of approximately 25% of patients with mild cognitive impairment (MCI) and 50% of those with AD, compared to individuals without dementia. RNA sequencing of plasma samples revealed decreased levels of carbohydrate sulfotransferase 3 (CHST3) mRNA, which is mainly expressed in oligodendrocyte (OLG)-lineage cells, in ECRG4-positive patients. Functional analyses demonstrated that the ECRG4 (71-107) peptide induced cytotoxicity in oligodendrocytes in vitro and reduced the expression of the OLG marker galactocerebroside in the corpus callosum following intracerebral injection. Additionally, peptide injection resulted in extravascular IgG leakage and the accumulation of OLG precursor cells (OPCs), which are crucial for myelin regeneration, around the blood vessels. These phenomena were similarly observed in the hippocampi of patients with EAD. Collectively, these findings establish ECRG4 as a novel serum marker for AD and suggest its association with ECRG4-dependent OLG dysfunction.

BackgroundAlzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by the accumulation of amyloid-β (Aβ) and the formation of neurofibrillary tangles. Although the amyloid cascade hypothesis underscores the centrality of Aβ accumulation, the precise initiators of this process remain unknown.ObjectiveIn this study, we investigate the potential role of Esophageal Cancer-Related Gene 4 (ECRG4) in AD. We hypothesized that ECRG4, which is associated with cognitive impairment and upregulated in AD, directly contributes to amyloid pathology.MethodsWe performed cell-based assays, co-immunoprecipitation, in vivo experiments using APPNL-G-F/NL-G-F knock-in mouse, and immunohistochemistry of human hippocampal sections.ResultsECRG4(133-148) associated with the amyloid precursor protein (APP) intracellular domain (AICD), leading to increased APP/Aβ accumulation. Furthermore, intracerebral injection of synthetic ECRG4(133-148) into AD model mice significantly augmented APP/Aβ deposition. Notably, the co-localization of ECRG4(133-148)-containing peptides with AICD-containing peptides increased with AD severity in human hippocampal tissue.ConclusionsOur findings establish that the carboxy-terminal fragment of ECRG4 acts as a potential initiator of amyloid pathology in AD through its interaction with AICD.

Esophageal cancer-related gene-4 (ECRG4) is a hormone-like protein that acts as a tumor suppressor by inducing cell death through apoptosis and cell-cycle arrest. ECRG4 is expressed in various organs, including the brain, and it influences the viability and function of neural cells such as oligodendrocytes and microglia. However, the effects of ECRG4 on astrocytes, which are essential neural cells that play significant roles in maintaining brain homeostasis, remain unclear. In this study, we demonstrate that ECRG4, specifically the region encompassing amino acids 71-107 (ECRG4(71-107)), induces cell death in mouse primary astrocytes. The ECRG4(71-107)-induced astrocyte death is associated with lysosomal damage and is mediated by cathepsins, indicating a lysosome-dependent cell death mechanism. Notably, ECRG4(71-107) promotes the secretion of cathepsins into the extracellular space. The introduction of exogenous cathepsins into the medium induced astrocyte death, while the removal of cathepsins from the conditioned medium reduced the toxicity associated with ECRG4 (71-107). These findings suggest that extracellular cathepsins mediate the toxicity of ECRG4(71-107) in astrocytes. Overall, this study reveals a novel mechanism of cell death induced by ECRG4 in astrocytes and suggests a potential involvement of ECRG4 in pathophysiological functions of the brain.

BackgroundSince glioblastoma (GBM)-initiating cells (GICs) were identified as the cells-of-origin for GBM, various GIC factors have been analyzed as potential therapeutic targets. However, these targets are also present in normal cells outside of the brain, raising concerns about potential side effects when directly targeted. The aim of this study is to develop a novel method that specifically eradicates GICs with reducing side effects.MethodsWe selected micoRNAs (miRs) that are significantly decreased in GICs compared to normal cells and developed a genome-editing (GE) system that knocks out a functional GIC factor in a miR-dependent manner (miR-dependent GE). Additionally, we developed mosaic-capsids that consist of braintropic and universal capsids, which deliver genes into GIC brain tumors.ResultsSystemic administration of the mosaic-capsids Adeno-associated virus (AAV) carrying a miR-dependent GFP expression cassette selectively expressed GFP in GICs transplanted into the brains of immunodeficient mice, without expression in either mouse brain cells or non-brain tissues. The mosaic-capsids AAV carrying a miR-dependent GE prevented GIC tumorigenesis in the brain and extended the survival time of tumor-bearing mice.ConclusionThese data indicate that the mosaic-capsids AAV containing a miR-dependent GE represents a novel therapeutic virus for GBM with fewer side effects.

AIMS:Epithelial V-like antigen 1 (Eva1) is a highly specific marker for brown adipose tissue (BAT) in both mice and humans, but its metabolic function remains unclear. We investigated the impact of Eva1 deletion on the development of obesity. METHODS:To assess the metabolic role of Eva1, we generated whole-body and adipocyte-specific Eva1knockout (KO) mice, which were subjected to a high-fat diet (HFD) for 12 weeks and characterized metabolic phenotypes. To further elucidate the depot-dependent impact of Eva1 deficiency, we performed histological analysis and 3' mRNA-seq of BAT and epididymal visceral white adipose tissue (eWAT). To investigate the role of macrophage-derived Eva1 in obesity development, we transplanted wild-type (WT) or Eva1KO macrophages into Eva1KO mice fed an HFD. RESULTS:We found that whole-body Eva1KO mice are resistant to HFD-induced obesity, insulin resistance and visceral adipose inflammation. However, Eva1 deletion in adipocytes, both brown and white, did not phenocopy these protective effects. Notably, whole-body Eva1 deficiency triggers functional changes in eWAT, but not in BAT. These results led us to investigate a possible involvement of macrophages in Eva1-mediated obesity regulation. We found that Eva1 is expressed in macrophages and plays a role in lipopolysaccharide (LPS)-induced inflammatory responses, possibly through the direct interaction with toll-like receptor 4 (TLR4). Moreover, Eva1KO mice exhibited improved survival rates in the face of severe sepsis induced by LPS. Importantly, transplantation of WT macrophages to Eva1KO mice abolished the beneficial effects of whole-body Eva1 deletion against obesity and visceral adipose inflammation. CONCLUSION:Our findings highlight macrophage-derived Eva1 as an important mediator in obesity-induced eWAT remodeling, suggesting that targeting Eva1 could offer a novel therapeutic strategy for obesity-related metabolic disorders.

Retinitis pigmentosa (RP) and macular dystrophy (MD) cause severe retinal dysfunction, affecting 1 in 4000 people worldwide. This disease is currently assumed to be intractable, because effective therapeutic methods have not been established, regardless of genetic or sporadic traits. Here, we examined a RP mouse model in which the Prominin-1 (Prom1) gene was deficient and investigated the molecular events occurring at the outset of retinal dysfunction. We extracted the Prom1-deficient retina subjected to light exposure for a short time, conducted single-cell expression profiling, and compared the gene expression with and without stimuli. We identified the cells and genes whose expression levels change directly in response to light stimuli. Among the genes altered by light stimulation, Igf1 was decreased in rod photoreceptor cells and astrocytes under the light-stimulated condition. Consistently, the insulin-like growth factor (IGF) signal was weakened in light-stimulated photoreceptor cells. The recovery of Igf1 expression with the adeno-associated virus (AAV) prevented photoreceptor cell death, and its treatment in combination with the endothelin receptor antagonist led to the blockade of abnormal glial activation and the promotion of glycolysis, thereby resulting in the improvement of retinal functions, as assayed by electroretinography. We additionally demonstrated that the attenuation of mammalian/mechanistic target of rapamycin (mTOR), which mediates IGF signalling, leads to complications in maintaining retinal homeostasis. Together, we propose that combinatorial manipulation of distinct mechanisms is useful for the maintenance of the retinal condition.

Cancer consists of heterogeneous cells, including cancer stem cells (CSCs), cancer cells, and tumor-associated cells, such as immune cells and vascular cells. Considering that these diverse cell types influence one another directly and indirectly through membrane proteins and secretion factors, such as exosomes and growth factors, the overall heterogeneity affects tumorigenicity and resistance to therapy. This review explores cancer heterogeneity, focusing on CSC heterogeneity, and discussed how the heterogeneity emerges by the intrinsic mechanism and the external factors and affects response to therapy. Additionally, as a potential therapeutic strategy to address this heterogeneity, I propose new Adeno-associated virus carrying a miRNA-dependent CSC eradication system that targets all types of CSCs with minimizing side effects.

Abstract PURPOSE The rise in aging population worldwide is increasing death from cancer, including glioblastoma. MicroRNAs (miRNAs/miRs) are small non-coding RNAs that mediate the posttranscriptional silencing of specific target mRNAs and that are currently recognized as important regulators of tumorigenesis and development. Here, we explore the impact of brain aging and aging-specific miRNAs on elderly glioblastomas. METHODS We comprehensively analyzed miRNAs expression using the TCGA dataset and identified miRNAs that are specifically expressed in elderly glioblastomas. To create the Ehime Glioma Genome Atlas (EGGA), RNA sequencing was performed using tumor tissues from 8 glioblastoma patients (4 elderly and 4 non-elderly). First, we evaluated the impact of the identified miRNAs on prognosis using TCGA and CGGA datasets and searched for target genes of specific miRNAs using TargetScan. The expression patterns of the identified target genes were verified using EGGA, TCGA, and CGGA datasets. RESULTS We divided the TCGA dataset into elderly and non-elderly groups based on three ages (75 years-old, 70 years-old, and 65 years-old) and analyzed the expression of miRNAs in tumor tissues. There were 3 miRNAs that showed abnormalities (1 increased miRNA, 2 decreased miRNAs), and among these, miR-210 (increased expression) and miR-507 (increased expression) were identified as miRNAs related to prognosis. Next, we analyzed the expression of EGGA and identified 410 mRNAs (368 increased mRNAs, 42 decreased mRNAs) that showed significantly abnormal expression in the elderly group. The target genes searched using TargetScan were 4,048 genes for miR-210 and 4,704 genes for miR-507. When compared with the 410 genes identified using EGGA, PLA2G3, a target gene to miR-507, was identified. The expression of PLA2G3 was also specifically increased in elderly glioblastoma in the TCGA dataset, and it had a strong influence on prognosis. CONCLUSION Our results found an aging-specific miRNA “miR-507” and its target gene “PLA2G3” that suggested as potential new therapeutic target molecules for improving the prognosis of elderly glioblastomas.

Background. The discovery of glioblastoma (GBM)-initiating cells (GICs) has impacted GBM research.These cells are not only tumorigenic but also exhibit resistance to radiotherapy and chemotherapy. Therefore, it is crucial to characterize GICs thoroughly and identify new therapeutic targets. In a previous study, we successfully identified epithelial-V-like antigen 1 (EVA1) as a novel functional factor specific to GICs. Methods. Hybridoma cells were generated by immunizing BALB/c mice with EVA1-Fc fusion protein. The reactivity of the supernatant from these hybridoma cells was examined using EVA1-overexpressing cells and GICs. Candidate antibodies were further selected using Biacore surface plasmon resonance analysis and 2 cytotoxicity assays-antibody-dependent cell cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Among the antibodies, the cytotoxicity of the B2E5-antibody drug conjugate (B2E5-ADC) was evaluated by both adding it to cultured GICs and injecting it into GIC tumor-bearing brains. Results. B2E5 demonstrated a high affinity for human EVA1 and effectively killed both EVA1-expressing cell lines and GICs in culture through ADCC and CDC. B2E5-ADC also exhibited strong cytotoxicity to GICs in culture and prevented their tumorigenesis in the brain when administered intracranially to the tumor-bearing brain. Conclusion. Our data indicate that B2E5-ADC is a new and promising therapeutic strategy for GBM. [GRAPHICS]

<p>Description of additional methods and procedures used in the study.</p>

Supplementary Figures S1-10 Supplementary Figure S1. Characterization of hGICs demonstrates the stemness nature of the cells. Supplementary Figure S2. miR-340 transfection causes a markedly elevated level of miR-340 expression in hGICs compared with the control. Supplementary Figure S3. miR-340 overexpression in glioma cell lines moderately inhibits tumorigenesis in vivo. Supplementary Figure S4. miR-340 overexpression affects the expression of the genes related to two signaling pathways. Supplementary Figure S5. Inhibition of PLAT expression by miR-340 overexpression in hGICs and co-immunolocalization of PLAT and CD15 in GBM tissues. Supplementary Figure S6. Knockdown efficiency of the PLAT-shRNA expression vector. Supplementary Figure S7. Effects of PLAT-shRNA are due to the specific inhibition of PLAT in hGICs. Supplementary Figure S8. Inhibitory effects of miR-340-overexpressing hGICs on in vitro cell functions are recovered by PLAT introduction. Supplementary Figure S9. Microarray analysis on the expression of c-Met, CD44, Sox2, DNMT1, ANXA2, and CDKN1A genes in hGICs by miR-340 overexpression. Supplementary Figure S10. Survival curves of glioma patients with different expression levels of plat.

Background The cancer stem cell theory proposes that tumor formation in vivo is driven only by specific tumor-initiating cells having stemness; however, clinical trials conducted to test drugs that target the tumor stemness provided unsatisfactory results thus far. Recent studies showed clear involvement of immunity in tumors; however, the requirements of tumor-initiation followed by stable growth in immunocompetent individuals remain largely unknown.Methods To clarify this, we used two similarly induced glioblastoma lines, 8B and 9G. They were both established by overexpression of an oncogenic H-RasL61 in p53-deficient neural stem cells. In immunocompromised animals in an orthotopic transplantation model using 1000 cells, both show tumor-forming potential. On the other hand, although in immunocompetent animals, 8B shows similar tumor-forming potential but that of 9G’s are very poor. This suggests that 8B cells are tumor-initiating cells in immunocompetent animals. Therefore, we hypothesized that the differences in the interaction properties of 8B and 9G with immune cells could be used to identify the factors responsible for its tumor forming potential in immunocompetent animals and performed analysis.Results Different from 9G, 8B cells induced senescence-like state of macrophages around tumors. We investigated the senescence-inducing factor of macrophages by 8B cells and found that it was interleukin 6. Such senescence-like macrophages produced Arginase-1, an immunosuppressive molecule known to contribute to T-cell hyporesponsiveness. The senescence-like macrophages highly expressed CD38, a nicotinamide adenine dinucleotide (NAD) glycohydrolase associated with NAD shortage in senescent cells. The addition of nicotinamide mononucleotide (NMN), an NAD precursor, in vitro inhibited to the induction of macrophage senescence-like phenotype and inhibited Arginase-1 expression resulting in retaining T-cell function. Moreover, exogenous in vivo administration of NMN after tumor inoculation inhibited tumor-initiation followed by stable growth in the immunocompetent mouse tumor model.Conclusions We identified one of the requirements for tumor-initiating cells in immunocompetent animals. In addition, we have shown that tumor growth can be inhibited by externally administered NMN against macrophage senescence-like state that occurs in the very early stages of tumor-initiating cell development. This therapy targeting the immunosuppressive environment formed by macrophage senescence-like state is expected to be a novel promising cancer therapeutic strategy.

Eva1 expression level affects the NF-κB activity that is indispensable for the proliferation of NSCL61.

Relationship between relb expression and the prognosis.

Isolation of colon CICs as SP cells and the expression of OR7C1

Supplementary Figure 6 from Sox11 Prevents Tumorigenesis of Glioma-Initiating Cells by Inducing Neuronal Differentiation

PDF file - 74K, RT-PCR analysis