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Abstract Background Extensive local invasion of glioblastoma (GBM) cells within the central nervous system (CNS) is one factor that severely limits current treatments. The aim of this study was to uncover genes involved in the invasion process, which could also serve as therapeutic targets. For the isolation of invasive GBM cells from non-invasive cells, we used a three-dimensional organotypic co-culture system where glioma stem cell (GSC) spheres were confronted with brain organoids (BOs). Using ultra-low input RNA sequencing (ui-RNA Seq), an invasive gene signature was obtained that was exploited in a therapeutic context. Methods GFP-labeled tumor cells were sorted from invasive and non-invasive regions within co-cultures. Ui-RNA sequencing analysis was performed to find a gene cluster up-regulated in the invasive compartment. This gene cluster was further analyzed using the Connectivity MAP (CMap) database. This led to the identification of SKF83566, an antagonist of the D1 dopamine receptor (DRD1), as a candidate therapeutic molecule. Knockdown and overexpression experiments were performed to find molecular pathways responsible for the therapeutic effects of SKF83566. Finally, the effects of SKF83566 were validated in orthotopic xenograft models in vivo. Results Ui-RNA seq analysis of three GSC cell models (P3, BG5 and BG7) yielded a set of 27 differentially expressed genes between invasive and non-invasive cells. Using CMap analysis, SKF83566 was identified as a selective inhibitor targeting both DRD1 and DRD5. In vitro studies demonstrated that SKF83566 inhibited tumor cell proliferation, GSC sphere formation, and invasion. RNA sequencing analysis of SKF83566-treated P3, BG5, BG7, and control cell populations yielded a total of 32 differentially expressed genes, that were predicted to be regulated by c-Myc. Of these, the UHRF1 gene emerged as the most downregulated gene following treatment, and ChIP experiments revealed that c-Myc binds to its promoter region. Finally, SKF83566, or stable DRD1 knockdown, inhibited the growth of orthotopic GSC (BG5) derived xenografts in nude mice. Conclusions DRD1 contributes to GBM invasion and progression by regulating c-Myc entry into the nucleus that affects the transcription of the UHRF1 gene. SKF83566 inhibits the transmembrane protein DRD1, and as such represents a candidate small therapeutic molecule for GBMs.

Abstract Background Valtrate, a natural compound isolated from the root of Valeriana, exhibits antitumor activity in many cancers through different mechanisms. However, its efficacy for the treatment of glioblastoma (GBM), a tumor type with a poor prognosis, has not yet been rigorously investigated. Methods GBM cell lines were treated with valtrate and CCK-8, colony formation and EdU assays, flow cytometry, and transwell, 3D tumor spheroid invasion and GBM-brain organoid co-culture invasion assays were performed to assess properties of proliferation, viability, apoptosis and invasion/migration. RNA sequencing analysis on valtrate-treated cells was performed to identify putative target genes underlying the antitumor activity of the drug in GBM cells. Western blot analysis, immunofluorescence and immunohistochemistry were performed to evaluate protein levels in valtrate-treated cell lines and in samples obtained from orthotopic xenografts. A specific activator of extracellular signal-regulated kinase (ERK) was used to identify the pathways mediating the effect. Results Valtrate significantly inhibited the proliferation of GBM cells in vitro by inducing mitochondrial apoptosis and suppressed invasion and migration of GBM cells by inhibiting levels of proteins associated with epithelial mesenchymal transition (EMT). RNA sequencing analysis of valtrate-treated GBM cells revealed platelet-derived growth factor receptor A (PDGFRA) as a potential target downregulated by the drug. Analysis of PDGFRA protein and downstream mediators demonstrated that valtrate inhibited PDGFRA/MEK/ERK signaling. Finally, treatment of tumor-bearing nude mice with valtrate led to decreased tumor volume (fivefold difference at day 28) and enhanced survival (day 27 vs day 36, control vs valtrate-treated) relative to controls. Conclusions Taken together, our study demonstrated that the natural product valtrate elicits antitumor activity in GBM cells through targeting PDGFRA and thus provides a candidate therapeutic compound for the treatment of GBM.

Abstract Background The tripartite motif (TRIM) family of proteins plays a key role in the developmental growth and therapeutic resistance of many tumors. However, the regulatory mechanisms and biological functions of TRIM proteins in human glioblastoma (GBM) are not yet fully understood. In this study, we focused on TRIM56, which emerged as the most differentially expressed TRIM family member with increased expression in GBM. Methods Western blot, real-time quantitative PCR (qRT-PCR), immunofluorescence (IF) and immunohistochemistry (IHC) were used to study the expression levels of TRIM56 and cIAP1 in GBM cell lines. Co-immunoprecipitation (co-IP) was used to explore the specific binding between target proteins and TRIM56. A xenograft animal model was used to verify the tumor promoting effect of TRIM56 on glioma in vivo. Results We observed elevated expression of TRIM56 in malignant gliomas and revealed that TRIM56 promoted glioma progression in vitro and in a GBM xenograft model in nude mice. Analysis of the Human Ubiquitin Array and co-IPs showed that cIAP1 is a protein downstream of TRIM56. TRIM56 deubiquitinated cIAP1, mainly through the zinc finger domain (amino acids 21–205) of TRIM56, thereby reducing the degradation of cIAP1 and thus increasing its expression. TRIM56 also showed prognostic significance in overall survival of glioma patients. Conclusions TRIM56-regulated post-translational modifications may contribute to glioma development through stabilization of cIAP1. Furthermore, TRIM56 may serve as a novel prognostic indicator and therapeutic molecular target for GBM.

Abstract Background Small peptides encoded by long non-coding RNAs (lncRNAs) have attracted attention for their various functions. Recent studies indicate that these small peptides participate in immune responses and antigen presentation. However, the significance of RNA modifications remains unclear. Methods Thirteen non-m6A-related neoantigen-coding lncRNAs were selected for analysis from the TransLnc database. Next, a neoantigen activation score (NAS) model was established based on the characteristics of the lncRNAs. Machine learning was employed to expand the model to two additional RNA-seq and two single-cell sequencing datasets for further validation. The DLpTCR algorithm was used to predict T cell receptor (TCR)-peptide binding probability. Results The non-m6A-related NAS model predicted patients’ overall survival outcomes more precisely than the m6A-related NAS model. Furthermore, the non-m6A-related NAS was positively correlated with tumor cells’ evolutionary level, immune infiltration, and antigen presentation. However, high NAS gliomas also showed more PD-L1 expression and high mutation frequencies of T-cell positive regulators. Interestingly, results of intercellular communication analysis suggest that T cell-high neoplastic cell interaction is weaker in both of the NAS groups which might arise from decreased IFNGR1 expression. Moreover, we identified unique TCR-peptide pairs present in all glioma samples based on peptides encoded by the 13 selected lncRNAs. And increased levels of neoantigen-active TCR patterns were found in high NAS gliomas. Conclusions Our work suggests that non-m6A-related neoantigen-coding lncRNAs play an essential role in glioma progression and that screened TCR clonotypes might provide potential avenues for chimeric antigen receptor T cell (CAR-T) therapy for gliomas.

BackgroundNitrosamines (NAms) are highly carcinogenic and frequently detected in drinking water systems in China, indicating potential human health risk through drinking water.ObjectiveTo analyze the distribution of NAms in drinking water in Shanghai and to evaluate relevant human health risk.MethodsA total of 94 samples of source water and 120 samples of finished water were collected in February (dry period) and August (wet period) of 2021 from 30 drinking water plants in Shanghai, and eight NAms were quantitatively analyzed by gas chromatography tandem mass spectrometry after solid phase extraction. Cancer risks for different age groups caused by NAms exposure through water were evaluated using Monte Carlo probabilistic method and carcinogens risk assessment model recommended by the United States Environmental Protection Agency.ResultsThe concentrations of total NAms in source and finished water were 12.56-65.86 ng·L−1 and 8.52-57.38 ng·L−1, respectively. The average concentration of total NAms was higher in source water (33.50 ng·L−1) than in finished water (22.07 ng·L−1, P

Resibufogenin (RB) is a major active ingredient in the traditional Chinese medicine Chansu and has garnered considerable attention for its efficacy in the treatment of cancer. However, the anticancer effects and underlying mechanisms of RB on glioblastoma (GBM) remain unknown. Here, we found that RB induced G2/M phase arrest and inhibited invasion in a primary GBM cell line, P3#GBM, and two GBM cell lines, U251 and A172. Subsequently, we demonstrated that RB-induced G2/M phase arrest occurred through downregulation of CDC25C and upregulation of p21, which was caused by activation of the MAPK/ERK pathway, and that RB inhibited GBM invasion by elevating intercellular Ca2+ to suppress the Src/FAK/Paxillin focal adhesion pathway. Intriguingly, we confirmed that upon RB binding to ATP1A1, Na+-K+-ATPase was activated as a receptor and then triggered the intracellular MAPK/ERK pathway and Ca2+-mediated Src/FAK/Paxillin focal adhesion pathway, which led to G2/M phase arrest and inhibited the invasion of GBM cells. Taken together, our findings reveal the antitumor mechanism of RB by targeting the ATP1A1 signaling cascade and two key signaling pathways and highlight the potential of RB as a new class of promising anticancer agents.