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Given that glioma stem cells (GSCs) play a critical role in the initiation and chemoresistance in glioblastoma multiforme (GBM), targeting GSCs is an attractive strategy to treat GBM. Utilizing an anti-cancer compound library, we identified R406, the active metabolite of a FDA-approved Syk inhibitor for immune thrombocytopenia (ITP), with remarkable cytotoxicity against GSCs but not normal neural stem cells. R406 significantly inhibited neurosphere formation and triggered apoptosis in GSCs. R406 induced a metabolic shift from glycolysis to oxidative phosphorylation (OXPHOS) and subsequently production of excess ROS in GSCs. R406 also diminished tumor growth and efficiently sensitized gliomas to temozolomide in GSC-initiating xenograft mouse models. Mechanistically, the anti-GSC effect of R406 was due to the disruption of Syk/PI3K signaling in Syk-positive GSCs and PI3K/Akt pathway in Syk-negative GSCs respectively. Overall, these findings not only identify R406 as a promising GSC-targeting agent but also reveal the important role of Syk and PI3K pathways in the regulation of energy metabolism in GSCs.

Lots of studies have demonstrated that anterior cingulate cortex plays important roles in the pain perception and pain modulation. The present study explored the role of mu-opioid receptor in nociceptive modulation in anterior cingulate cortex of rats with neuropathic pain. Neuropathic pain model was set up by chronic constriction injury of the left sciatic nerve of rats. The hindpaw withdrawal latency to thermal and mechanical stimulation, by hot plate and Randall Selitto Test respectively, was used to evaluate the rat’s responses to noxious stimulation. Results showed that intra-anterior cingulate cortex injection of morphine could induce the antinociception dose-dependently. By intra-anterior cingulate cortex injection of opioid receptor antagonist, the morphine-induced antinociception could be attenuated by naloxone, as well as much significantly by the selective mu-opioid receptor antagonist β-funaltrexamine, indicating that mu-opioid receptor is involved in the morphine-induced antinociception in anterior cingulate cortex of rats with neuropathic pain. The morphine-induced antinociception was much more decreased in rats with neuropathic pain than that in normal rats, and there was a significant decrease in mu-opioid receptor messenger RNA levels in anterior cingulate cortex of rats with neuropathic pain, indicating that there may be a down-regulation in mu-opioid receptor expression in anterior cingulate cortex of rats with neuropathic pain. To further confirm the role of mu-opioid receptor in morphine-induced antinociception in anterior cingulate cortex, normal rats were received intra-anterior cingulate cortex administration of small interfering RNA targeting mu-opioid receptor and it was found that there was a down-regulation in mu-opioid receptor messenger RNA levels, as well as a down-regulation in mu-opioid receptor expression in anterior cingulate cortex tested by real-time polymerase chain reaction and western blotting. Furthermore, the morphine-induced antinociceptive effect decreased significantly in rats with small interfering RNA targeting mu-opioid receptor, which indicated that knockdown mu-opioid receptor in anterior cingulate cortex could also attenuate morphine-induced antinociceptive effect. These results strongly suggest that mu-opioid receptor plays a significant role in nociceptive modulation in anterior cingulate cortex of rats.

Background A profound understanding of the molecular landscape of glioblastoma multiforme (GBM) will make it possible to develop better and more intelligent therapies directed toward specific molecular targets and may one day yield better prognostic capabilities. Immune checkpoint molecules have inspired the emergence of immune checkpoint-targeting therapeutic strategies. However, the prognostic significance of the immune checkpoint molecule T cell immunoglobulin mucin-3 (Tim-3) on tumor-infiltrating immune cells (TIICs) and O-6-methylguanine-DNA methyltransferase (MGMT) promoter methylation status has not yet been fully elucidated. We aimed to develop an MGMT promoter methylation status-associated immune prognostic signature for GBM. Patients and methods A total of 84 patients with newly diagnosed GBM were included in this study. MGMT promoter methylation status was retrospectively analyzed, and the expression level of Tim-3 was investigated using immunohistochemistry (IHC). The correlation between Tim-3 expression combined with MGMT promoter methylation status and prognosis was explored. Results Tim-3 expression varied in GBM patients. Mesenchymal expression of Tim-3 in GBM tissues was present 73.81% (62/84) of patients, and these were subdivided into groups based on low 15.48% (13/84), moderate 7.14% (6/84), or strong expression 51.19% (43/84). Forty-eight patients had tumors that tested positive for MGMT promoter methylation, while the remaining 36 patients tested negative. Conclusions We profiled the immune status of MGMT promoter methylation in GBM and established a local immune signature for GBM that could independently identify patients with a favorable prognosis, indicating a relationship between prognosis and GBM immune signature. MGMT promoter methylation with lower Tim-3 expression was significantly associated with better survival.

It is known that calcitonin gene-related peptide (CGRP) plays a key role in pain modulation in the brain. There are high expressions of CGRP and CGRP receptor in anterior cingulate cortex (ACC), an important brain structure in pain modulation. The present study explored the role and mechanisms of CGRP and CGRP receptor in nociceptive modulation in ACC in naïve rats and inflammatory rats. Administration of different does of CGRP in ACC induced significant antinociception in a dose-dependent manner in both naïve rats and rats with inflammatory pain. The CGRP-induced antinociception was attenuated by injection of the CGRP receptor antagonist CGRP8-37 in ACC. Interestingly, both CGRP-induced antinociception and CGRP receptor expression decreased in ACC in rats with inflammatory pain compared with naïve rats. Knockdown of CGRP receptor in ACC by siRNA targeting to CGRP receptor attenuated both the CGRP receptor expression and the CGRP-induced antinociception significantly in rats. These findings demonstrate that CGRP and CGRP receptor participate in nociceptive modulation in ACC in rats, inhibiting CGRP receptor expression induces decrease in CGRP-induced antinociception in ACC.

Background The treatment options for newly diagnosed non–small-cell lung cancer (NSCLC) patients with 1 to 3 synchronous brain metastases (BM) remain controversial. The current study aimed to comprehensively analyze the characteristics, local treatment paradigms, and survival outcomes in these populations. Patients and Methods A total of 252 NSCLC patients initially diagnosed with 1 to 3 synchronous brain-only metastases were enrolled onto this study. Local therapy (LT) to primary lung tumors (PLT) and BM included surgery, radiotherapy, or both. Median overall survival (mOS) was measured among patients who received LT to both PLT and BM (all-LT group), patients who were treated with LT to either PLT or BM (part-LT group), and patients who did not receive any LT (non-LT group). Results The mOS for all-LT (n = 70), part-LT (n = 113), and non-LT (n = 69) groups was 33.2, 18.5, and 16.8 months, respectively (P = .002). The OS rates at 5 years for the all-LT, part-LT, and non-LT groups were 25.5%, 16.2%, and 0, respectively. Multivariable analysis revealed that all-LT versus non-LT, pretreatment Karnofsky performance status > 70, histology of adenocarcinoma, thoracic stage I-II, EGFR mutation, ALK positive, and second-line systemic therapies were independent prognostic factors for improved mOS. Conclusions The current study showed that LT for both PLT and BM is associated with superior OS in appropriately selected NSCLC patients initially diagnosed with 1 to 3 synchronous BM. Prospective trials are urgently needed to confirm this finding.

Prolyl 4-Hydroxylase Subunit Alpha 1 (P4HA1) is the active catalytic component of prolyl 4-hydroxylase and plays a crucial role in modulating extracellular matrix hemostasis. P4HA1 has been reported to promote tumor progression by enhancing invasion and angiogenesis. Overexpression of P4HA1 is associated with decreased survival for patients with breast and prostate cancer. However, the prognostic significance of P4HA1 for glioma patients remains undefined.The expression of P4HA1 in 290 gliomas (WHO grade II-IV) and 10 normal brain tissues was examined with TMA-based immunohistochemistry assay. The correlation between P4HA1 expression and clinicopathological parameters as well as the prognosis of glioma patients was investigated.Cytoplasmic expression of P4HA1 is high in 37.93% of all glioma cases, with 44.98% in high-grade gliomas and 19.75% in low-grade gliomas respectively. Increased P4HA1 level was correlated with advanced histological grade (p0.01) and old age (p=0.01). Upregulation of P4HA1, as well as histological grade, was an independent risk factor for unfavorable prognosis. Subgroup analysis demonstrated that high P4HA1 expression was significantly associated with poor prognosis for high-grade gliomas (p0.01) but not for low-grade gliomas.P4HA1 was upregulated in gliomas. High expression of P4HA1 was correlated with the malignancy of gliomas and could serve as a prognostic indicator for patients with high-grade gliomas.

Background: The latest WHO classification of CNS tumors using the integrated phenotypic and molecular parameters (IDH, ATRX, 1p19q, TERT etc.) have reestablished the CNS tumors classification in addition to traditional histology. The establishment of glioma molecular typing can more accurately predict prognosis, better guide individualized treatment to improve survival. Methods: The expression of IDH1, ATRX, PHH3, P53 and Ki67 was detected by IHC. Molecular status of IDH1/2 and TERT were analyzed using Sanger sequencing. MGMT was explored using methylation-specific PCR. 1p/19q codeletion status was firstly detected by FISH, then further confirmed by multiplex PCR-based next generation sequencing. Results: The mutation frequency of IDH1 was 68.7% (79/115) in WHO II astrocytoma, and 82 cases (82/344, 23.8%) were "triple-negative glioma" in our cohort. Multivariate COX analysis revealed that only IDH, 1p/19q, TERT and MGMT were independent prognostic factors. Noteworthily, we found 7 cases of the new molecular phenotype presented as "IDH wildtype and 1p/19q codeletion", not mentioned in the latest WHO guideline. Conclusion: We detected the newly recommended markers in a large cohort of Chinese glioma patients. Our data demonstrated a relatively lower frequency of IDH mutations and a higher prevalence of triple-negative glioma in Chinese compared with American and European, indicating ethnic and geographical difference in some markers. In addition, the new molecular phenotype "IDH wildtype and 1p/19q codeletion" glioma deserved special focus. These findings suggest that further stratification of infiltrating gliomas is needed for different treatment strategy and precision medicine.

Chromatinolysis refers to enzymatic degradation of nuclear DNA and is regarded as one of the crucial events leading to cell death. Mixed-lineage kinase domain-like protein (MLKL) has been identified as a key executor of necroptosis, but it remains unclear whether MLKL contributes to necroptosis via regulation of chromatinolysis. In this study, we find that shikonin induces MLKL activation and chromatinolysis in glioma cells in vitro and in vivo, which are accompanied with nuclear translocation of AIF and γ-H2AX formation. In vitro studies reveal that inhibition of MLKL with its specific inhibitor NSA or knockdown of MLKL with siRNA abrogates shikonin-induced glioma cell necroptosis, as well as chromatinolysis. Mechanistically, activated MLKL targets mitochondria and triggers excessive generation of mitochondrial superoxide, which promotes AIF translocation into nucleus via causing mitochondrial depolarization and aggravates γ-H2AX formation via improving intracellular accumulation of ROS. Inhibition of nuclear level of AIF by knockdown of AIF with siRNA or mitigation of γ-H2AX formation by suppressing ROS with antioxidant NAC effectively prevents shikonin-induced chromatinolysis. Then, we found that RIP3 accounts for shikonin-induced activation of MLKL, and activated MLKL reversely up-regulates the protein level of CYLD and promotes the activation of RIP1 and RIP3. Taken together, our data suggest that MLKL contributes to shikonin-induced glioma cell necroptosis via promotion of chromatinolysis, and shikonin induces a positive feedback between MLKL and its upstream signals RIP1 and RIP3.

Background Ependymal tumors are pathologically defined intrinsic neoplasms originating in the intracranial compartments or the spinal cord that affect both children and adults. The recently integrated classification of ependymomas based on both histological and molecular characteristics is capable of subgrouping patients with various prognoses. However, the application of histological and molecular markers in Chinese patients with ependymomas has rarely been reported. We aimed to demonstrate the significance of histological characteristics, the v-relavian reticuloendotheliosis viral oncogene homolog A (RELA) fusions and other molecular features in ependymal tumors. Methods We reviewed the histological characteristics of ependymal tumors using conventional pathological slides and investigate the RELA fusions and Cylclin D1 (CCND1) amplification by Fluorescence in situ hybridization (FISH) and trimethylation of histone 3 lysine 27 (H3K27me3) expression by immunohistochemistry (IHC) methods. SPSS software was used to analyze the data. Results We demonstrated that hypercellularity, atypia, microvascular proliferation, necrosis, mitosis, and an elevated Ki-67 index, were tightly associated with an advanced tumor grade. Tumor location, necrosis, mitosis and the Ki-67 index were related to the survival of the ependymomas, but Ki67 was the only independent prognostic factor. Additionally, RELA fusions, mostly presented in pediatric grade III intracranial ependymomas, indicated decreased survival times of patients, and closely related to the patients’ age, tumor grade, cellularity, cellular atypia, necrosis and Ki67 index in the intracranial ependymal tumors, whereas reduction of H3K27me3 predicted the worse prognosis in ependymal tumors. Conclusions Histological and molecular features facilitate tumor grading and prognostic predictions for ependymal tumors in Chinese patients.

Activation of the cyclic adenosine monophosphate/protein kinase A (cAMP/PKA) pathway induces glial differentiation of glioblastoma (GBM) cells, but the mechanism by which microRNA (miRNA) regulate this process remains poorly understood. In this study, by performing miRNA genomics and loss- and gain-of-function assays in dibutyryl-cAMP-treated GBM cells, we identified a critical negative regulator, hsa-miR-1275, that modulates a set of genes involved in cancer progression, stem cell maintenance, and cell maturation and differentiation. Additionally, we confirmed that miR-1275 directly and negatively regulates the protein expression of glial fibrillary acidic protein (GFAP), a marker of mature astrocytes. Of note, tri-methyl-histone H3 (Lys27) (H3K27me3), downstream of the PKA/polycomb repressive complex 2 (PRC2) pathway, accounts for the downregulation of miR-1275. Furthermore, decreased miR-1275 expression and induction of GFAP expression were also observed in dibutyryl-cAMP-treated primary cultured GBM cells. In a patient-derived glioma stem cell tumor model, a cAMP elevator and an inhibitor of H3K27me3 methyltransferase inhibited tumor growth, induced differentiation, and reduced expression of miR-1275. In summary, our study shows that epigenetic inhibition of miR-1275 by the cAMP/PKA/PRC2/H3K27me3 pathway mediates glial induction of GBM cells, providing a new mechanism and novel targets for differentiation-inducing therapy.

Glioma is the most frequent primary central nervous system tumor. Although the current first-line medicine, temozolomide (TMZ), promotes patient survival, drug resistance develops easily. Thus, it is important to investigate novel therapeutic reagents to solidify the treatment effect. β-Elemene (bELE) is a compound from a Chinese herb whose anticancer effect has been shown in various types of cancer. However, its role in the inhibition of glioma stem-like cells (GSLCs) has not yet been reported. We studied both the in vitro and the in vivo inhibitory effect of bELE and TMZ in GSLCs and parental cells and their combined effects. The molecular mechanisms were also investigated. We also optimized the delivery methods of bELE. We found that bELE selectively inhibits the proliferation and sphere formation of GSLCs, other than parental glioma cells, and TMZ exerts its effects on parental cells instead of GSLCs. The in vivo data confirmed that the combination of bELE and TMZ worked better in the xenografts of GSLCs, mimicking the situation of tumorigenesis of human cancer. Notch1 was downregulated with bELE treatment. Our data also demonstrated that the continuous administration of bELE produces an ideal effect to control tumor progression. Our findings have demonstrated, for the first time, that bELE could compensate for TMZ to kill both GSLCs and nonstem-like cancer cells, probably improving the prognosis of glioma patients tremendously. Notch1 might be a downstream target of bELE. Therefore, our data shed light on improving the outcomes of glioma patients by combining bELE and TMZ.

Hao Duan,1,* Zhen-Qiang He,1,* Cheng-Cheng Guo,1 Jue-Hui Li,1 Jian Wang,1 Zhe Zhu,2 Ke Sai,1 Zhong-Ping Chen,1 Xiao-Bing Jiang,1 Yong-Gao Mou1 1Department of Neurosurgery/Neuro-Oncology, Sun Yat-sen University Cancer Center, State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine, Guangzhou, China; 2Department of Medicine, Division of Regenerative Medicine, University of California, San Diego, School of Medicine, La Jolla, CA, USA *These authors contributed equally to this work Purpose: The presence of brain metastasis (BM) in patients with colorectal cancer (CRC) is usually associated with terminal-stage illness; however, a subgroup of patients receiving aggressive treatment can have a satisfactory prognosis. This study was designed to investigate the profile of prognostic factors in CRC patients with BM treated aggressively. Patients and methods: CRC patients with BM were retrospectively reviewed. Survival analysis was performed to identify potential prognostic factors in the entire cohort of patients and a subgroup of patients treated aggressively. Aggressive treatments included surgical resection, radiotherapy, and/or chemotherapy. Overall survival was defined as the time between the diagnosis of BM and death or until the date of the last follow-up visit. Results: A total of 78 CRC patients were confirmed as having BM. Sixty-eight of them had extracranial metastases at the time of their BM diagnosis. The most common sites of extracranial metastases were lung (n=51, 65.4%), followed by liver (n=25, 32.1%) and bone (n=12, 15.4%). Fifty-one patients who were treated aggressively had significantly longer overall survival than those who accepted palliative care (14.1 months vs 2.0 months, P