Proteome, Lysine Acetylome, and Succinylome Identify Posttranslational Modification of STAT1 as a Novel Drug Target in Silicosis.

Inhalation of crystalline silica dust induces incurable lung damage, silicosis, and pulmonary fibrosis. However, the mechanisms of the lung injury remain poorly understood, with limited therapeutic options aside from lung transplantation. Posttranslational modifications can regulate the function of proteins and play an important role in studying disease mechanisms. To investigate changes in posttranslational modifications of proteins in silicosis, combined quantitative proteome, acetylome, and succinylome analyses were performed with lung tissues from silica-injured and healthy mice using liquid chromatography-mass spectrometry. Combined analysis was applied to the three omics datasets to construct a protein landscape. The acetylation and succinylation of the key transcription factor STAT1 were found to play important roles in the silica-induced pathophysiological changes. Modulating the acetylation level of STAT1 with geranylgeranylacetone effectively inhibited the progression of silicosis. This report revealed a comprehensive landscape of posttranslational modifications in silica-injured mouse and presented a novel therapeutic strategy targeting the posttranslational level for silica-induced lung diseases.

Inhibition of gasdermin D-dependent pyroptosis attenuates the progression of silica-induced pulmonary inflammation and fibrosis.

Silicosis is a leading cause of occupational disease-related morbidity and mortality worldwide, but the molecular basis underlying its development remains unclear. An accumulating body of evidence supports gasdermin D (GSDMD)-mediated pyroptosis as a key component in the development of various pulmonary diseases. However, there is little experimental evidence connecting silicosis and GSDMD-driven pyroptosis. In this work, we investigated the role of GSDMD-mediated pyroptosis in silicosis. Single-cell RNA sequencing of healthy and silicosis human and murine lung tissues indicated that GSDMD-induced pyroptosis in macrophages was relevant to silicosis progression. Through microscopy we then observed morphological alterations of pyroptosis in macrophages treated with silica. Measurement of interleukin-1ß release, lactic dehydrogenase activity, and real-time propidium iodide staining further revealed that silica induced pyroptosis of macrophages. Additionally, we verified that both canonical (caspase-1-mediated) and non-canonical (caspase-4/5/11-mediated) signaling pathways mediated silica-induced pyroptosis activation, in vivo and in vitro. Notably, Gsdmd knockout mice exhibited dramatically alleviated silicosis phenotypes, which highlighted the pivotal role of pyroptosis in this disease. Taken together, our results demonstrated that macrophages underwent GSDMD-dependent pyroptosis in silicosis and inhibition of this process could serve as a viable clinical strategy for mitigating silicosis.

Tetrandrine alleviates silicosis by inhibiting canonical and non-canonical NLRP3 inflammasome activation in lung macrophages.

Silicosis caused by inhalation of silica particles leads to more than ten thousand new occupational exposure-related deaths yearly. Exacerbating this issue, there are currently few drugs reported to effectively treat silicosis. Tetrandrine is the only drug approved for silicosis treatment in China, and despite more than decades of use, its efficacy and mechanisms of action remain largely unknown. Here, in this study, we established silicosis mouse models to investigate the effectiveness of tetrandrine of early and late therapeutic administration. To this end, we used multiple cardiopulmonary function test, as well as markers for inflammation and fibrosis. Moreover, using single cell RNA sequencing and transcriptomics of lung tissue and quantitative microarray analysis of serum from silicosis and control mice, our results provide a novel description of the target pathways for tetrandrine. Specifically, we found that tetrandrine attenuated silicosis by inhibiting both the canonical and non-canonical NLRP3 inflammasome pathways in lung macrophages. Taken together, our work showed that tetrandrine yielded promising results against silicosis-associated inflammation and fibrosis and further lied the groundwork for understanding its molecular targets. Our results also facilitated the wider adoption and development of tetrandirne, potentially accelerating a globally accepted therapeutic strategy for silicosis.

Pro-inflammatory and proliferative microglia drive progression of glioblastoma.

Scant understanding of the glioblastoma microenvironment and molecular bases hampers development of efficient treatment strategies. Analyses of gene signatures of human gliomas demonstrate that the SETD2 mutation is correlated with poor prognosis of IDH1/2 wild-type (IDH-WT) adult glioblastoma patients. To better understand the crosstalk between SETD2 mutant (SETD2-mut) glioblastoma cells and the tumor microenvironment, we leverage single-cell transcriptomics to comprehensively map cellular populations in glioblastoma. In this study, we identify a specific subtype of high-grade glioma-associated microglia (HGG-AM). Further analysis shows that transforming growth factor (TGF)-ß1 derived from SETD2-mut/IDH-WT tumor cells activates HGG-AM, exhibiting pro-inflammation and proliferation signatures. Particularly, HGG-AM secretes interleukin (IL)-1ß via the apolipoprotein E (ApoE)-mediated NLRP1 inflammasome, thereby promoting tumor progression. HGG-AM present extensive proliferation and infiltration to supplement the activated microglia pool. Notably, TGF-ß1/TßRI depletion dramatically reduces HGG-AM density and suppresses tumor growth. Altogether, our studies identify a specific microglia subpopulation and establish the cellular basis of interactions between HGG-AM and glioblastoma cells.

Necroptotic astrocytes contribute to maintaining stemness of disseminated medulloblastoma through CCL2 secretion.

BACKGROUND: Medulloblastoma (MB) with metastases at diagnosis and recurrence correlates with poor prognosis. Unfortunately, the molecular mechanism underlying metastases growth has received less attention than primary therapy-naïve MB. Though astrocytes have been frequently detected in brain tumors, their roles in regulating the stemness properties of MB stem-like cells (MBSCs) in disseminated lesions remain elusive. METHODS: Effects of tumor-associated astrocyte (TAA)-secreted chemokine C-C ligand 2 (CCL2) on MBSC self-renewal was determined by immunostaining analysis. Necroptosis of TAA was examined by measuring necrosome activity. Alterations in Notch signaling were examined after inhibition of CCL2. Progression of MBSC-derived tumors was evaluated after pharmaceutical blockage of necroptosis. RESULTS: TAA, as the essential components of disseminated tumor, produced high levels of CCL2 to shape the inflammation microenvironment, which stimulated the enrichment of MBSCs in disseminated MB. In particular, CCL2 played a pivotal role in maintaining stem-like properties via Janus kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3)-mediated activation of Notch signaling. Loss of CCL2/C-C chemokine receptor 2 (CCR2) function repressed the JAK2/STAT3-Notch pathway and impaired MBSC proliferation, leading to a dramatic reduction of stemness, tumorigenicity, and metastasizing capability. Furthermore, necroptosis-induced CCL2 release depended on activation of receptor-interacting protein 1 (RIP1)/RIP3/mixed lineage kinase domain-like pseudokinase (MLKL) in TAA, which promoted the oncogenic phenotype. Blockade of necroptosis resulted in CCL2 deprivation and compromised MBSC self-proliferation, indicating MBSCs outsourced CCL2 from necroptotic TAA. Finally, CCL2 was upregulated in high-risk stages of MB, further supporting its value as a prognostic indicator. CONCLUSION: These findings highlighted the critical role of CCL2/CCR2 in Notch signaling activation in MBSCs and revealed a necroptosis-associated glial cytokine microenvironment driving stemness maintenance in disseminations.Key Points1. TAA-derived CCL2 promoted stemness in disseminated MBSCs through Notch signaling activation via the JAK2/STAT3 pathway.2. TAA released CCL2 in a RIP1/RIP3/MLKL-dependent manner leading to necroptosis.

Neuraxis Metastases Of Primary Central Nervous System Tumors: A Review Of Clinicopathological And Radiographic Characters Of 198 Cases In A Single Center.

BACKGROUND: Neuraxis metastases (NM) from systemic and central nervous system (CNS) tumors have become increasingly common. However, a lack of systematic information restricts the development of the accurate diagnosis and treatment. The aim of this study is to facilitate the understanding of NM arising from CNS tumors in the largest cohort. METHODS: Based on the clinicopathological and neuroimaging findings, we retrospectively analyze the epidemiological characters, radiographic classification, therapeutic strategies and prognostic factors. RESULTS: A total of 198 cases are enrolled and the most common primary tumor is medulloblastoma (34.34%). The median age is 15.0 years and the majority of NM (79.29%) occur in the children and young adult groups. One hundred and forty-nine (75.25%) cases suffer from intracranial metastases, and 169 (85.35%) have intraspinal NM. The whole leptomeninges and cauda equine are the most preferential disseminated sites. Upon MRI parameters, the massive and miliary subgroup occurs most frequently in the intracranial and intraspinal NM, respectively. Treatment includes surgery (21.71%), chemotherapy alone (19.19%), radiation alone (10.10%) and combined therapy (55.56%). Operations are performed in order to identify pathology and relive masses, as well as the triple chemotherapeutic scheme consisting of ifosfamide, carboplatin and etoposide is recommended for most of NM. The median overall survival is 11.6 months. Younger age, coexistence of NM with primary tumors, shorter interval from primaries to metastases, glioma, leptomeningeal seeding and nodal subtype on MRI significantly correlate with poor prognosis. CONCLUSION: In spite of controversial therapies and poor outcomes, the neuroimaging classification and comprehensive treatment contribute to the efficient administration of NM.

EZH2 Phosphorylation Promotes Self-Renewal of Glioma Stem-Like Cells Through NF-κB Methylation.

Cancer stem-like cells (CSCs) is a cell population in glioma with capacity of self-renewal and is critical in glioma tumorigenesis. Parallels between CSCs and normal stem cells suggest that CSCs give rise to tumors. Oncogenic roles of maternal embryonic leucine-zipper kinase (MELK) and enhancer of zeste homolog 2 (EZH2) have been reported to play a crucial role in glioma tumorigenesis. Herein, we focus on mechanistic contributions of downstream molecules to maintaining stemness of glioma stem-like cells (GSCs). Transcriptional factor, NF-κB, co-locates with MELK/EZH2 complex. Clinically, we observe that the proportion of MELK/EZH2/NF-κB complex is elevated in high-grade gliomas, which is associated with poor prognosis in patients and correlates negatively with survival. We describe the interaction between these three proteins. Specifically, MELK induces EZH2 phosphorylation, which subsequently binds to and methylates NF-κB, leading to tumor proliferation and persistence of stemness. Furthermore, the interaction between MELK/EZH2 complex and NF-κB preferentially occurs in GSCs compared with non-stem-like tumor cells. Conversely, loss of this signaling dramatically suppresses the self-renewal capability of GSCs. In conclusion, our findings suggest that the GSCs depend on EZH2 phosphorylation to maintain the immature status and promote self-proliferation through NF-κB methylation, and represent a novel therapeutic target in this difficult to treat malignancy.

MELK and EZH2 Cooperate to Regulate Medulloblastoma Cancer Stem-like Cell Proliferation and Differentiation.

Medulloblastoma is the most common malignant brain tumor in children. Although accumulated research has suggested that cancer stem-like cells play a key role in medulloblastoma tumorigenesis, the specific molecular mechanism regarding proliferation remains elusive. Here, we reported more abundant expression of maternal embryonic leucine-zipper kinase (MELK) and enhancer of zeste homolog 2 (EZH2) in medulloblastoma stem-like cells than in neural stem cells and the interaction between the two proteins could mediate the self-renewal of sonic hedgehog subtype medulloblastoma. In human medulloblastoma, extensive nodularity and large-cell/anaplastic subgroups differed according to the staining levels of MELK and EZH2 from the other two subgroups. The proportion of MELK- or EZH2-positive staining status could be considered as a potential indicator for survival. Mechanistically, MELK bound to and phosphorylated EZH2, and its methylation was induced by EZH2 in medulloblastoma, which could regulate the proliferation of cancer stem-like cells. In xenografts, loss of MELK or EZH2 attenuated medulloblastoma stem-like cell-derived tumor growth and promoted differentiation. These findings indicate that MELK-induced phosphorylation and EZH2-mediated methylation in MELK/EZH2 pathway are essential for medulloblastoma stem-like cell-derived tumor proliferation, thereby identifying a potential therapeutic strategy for these patients.Implications: This study demonstrates that the interaction occurring between MELK and EZH2 promotes self-proliferation and stemness, thus representing an attractive therapeutic target and potential candidate for diagnosis of medulloblastoma. Mol Cancer Res; 15(9); 1275-86. ©2017 AACR.

Differential expression of folate receptor 1 in medulloblastoma and the correlation with clinicopathological characters and target therapeutic potential.

Medulloblastoma is the most common malignant brain tumor in children. Folate receptor 1 (Folr1) was abundantly expressed in some epithelial malignancies. However the expression profile and the role of clinicopathological significance and therapeutic target potential in medulloblastoma still remain elusive. Currently we detected the expression of Folr1 in medulloblastoma and identified the diagnostic application by evaluating the clinical, pathological and neuroimaging values. Then we developed a target therapeutic compound with Folr1, which exhibited promising efficiency in treatment of medulloblastoma. Folr1 expression was up-regulated in medulloblastoma and positively correlated with percentage of Ki-67 and MMP9 labeling, pathological subtypes, serum Folr1 levels and CSF spreading on MRI. The level of serum Folr1 showed rational sensitivity and specificity in predicting histological subgroups. Strong Folr1 expression was recommended as the independent value regarding the prognosis of patients with medulloblastoma. Folr1 targeted therapy attenuated the tumor growth and metastasis with down-regulation of MMPs proteins and activation of apoptosis. Immunostaining analysis in the xenograft samples showed the decreased Ki-67 and MMP9 index providing the strong evidences that Folr1 targeted application can suppress the proliferation and invasion. Our findings uncovered in Folr1 a predictive candidate and therapeutic target for medulloblastoma.