Research progress in molecular pathology markers in medulloblastoma.

Medulloblastoma (MB) is the commonest primary malignant brain cancer. The current treatment of MB is usually surgical resection combined with radiotherapy or chemotherapy. Although great progress has been made in the clinical management of MB, tumor metastasis and recurrence are still the main cause of death. Therefore, definitive and timely diagnosis is of great importance for improving therapeutic effects on MB. In 2016, the World Health Organization (WHO) divided MB into four subtypes: wingless-type mouse mammary tumor virus integration site (WNT), sonic hedgehog (SHH), non-WNT/non-SHH group 3, and group 4. Each subtype of MB has a unique profile in copy number variation, DNA alteration, gene transcription, or post-transcriptional/translational modification, all of which are associated with different biological manifestations, clinical features, and prognosis. This article reviewed the research progress of different molecular pathology markers in MB and summarized some targeted drugs against these molecular markers, hoping to stimulate the clinical application of these molecular markers in the classification, diagnosis, and treatment of MB.

Sitagliptin inhibits the survival, stemness and autophagy of glioma cells, and enhances temozolomide cytotoxicity.

The standard regimen treatment has improved GBM outcomes, but the survival rate of patients is still unsatisfactory. Temozolomide (TMZ) resistance is one of main reasons limiting the therapeutic efficacy of GBM. However, there are currently no TMZ-sensitizing drugs available in the clinic. Here we aimed to study whether the antidiabetic drug Sitagliptin can inhibit the survival, stemness and autophagy of GBM cells, and thus enhance TMZ cytotoxicity. We used CCK-8, EdU, colony formation, TUNEL and flow cytometry assays to assess cell proliferation and apoptosis; sphere formation and limiting dilution assays to measure self-renewal and stemness of glioma stem cells (GSCs); Western blot, qRT-PCR or immunohistochemical analysis to measure the expression of proliferation or stem cell markers; Western blot/fluorescent analysis of LC3 and other molecules to evaluate autophagy formation and degradation in glioma cells. We found that Sitagliptin inhibited proliferation and induced apoptosis in GBM cells and suppressed self-renewal and stemness of GSCs. The in vitro findings were further confirmed in glioma intracranial xenograft models. Sitagliptin administration prolonged the survival time of tumor-bearing mice. Sitagliptin could inhibit TMZ-induced protective autophagy and enhance the cytotoxicity of TMZ in glioma cells. In addition, Sitagliptin acted as a dipeptidyl peptidase 4 inhibitor in glioma as well as in diabetes, but it did not affect the blood glucose level and body weight of mice. These findings suggest that Sitagliptin with established pharmacologic and safety profiles could be repurposed as an antiglioma drug to overcome TMZ resistance, providing a new option for GBM therapy.

Effects of the m6Am methyltransferase PCIF1 on cell proliferation and survival in gliomas.

BACKGROUND: Previous studies have suggested an important role for N6-methyladenosine (m6A) modification in the proliferation of glioma cells. N6, 2'-O-dimethyladenosine (m6Am) is another methylated form affecting the fate and function of most RNA. PCIF1 has recently been identified as the sole m6Am methyltransferase in mammalian mRNA. However, it remains unknown about the role of PCIF1 in the growth and survival of glioma cells. METHODS: We constructed glioma cell lines that stably downregulated/upregulated PCIF1, established intracranial xenograft models using these cell lines, and employed the following methods for investigations: CCK-8, EdU, colony formation, flow cytometry, qRT-PCR, Western blot, and immunohistochemistry. FINDINGS: Downregulating PCIF1 promoted glioma cell proliferation, while overexpressing PCIF1 showed the opposite effects. Overexpression of PCIF1 blocked cell cycle progression and induced apoptosis in glioma cells, which was further confirmed by alterations in the expression of cell checkpoint proteins and apoptotic markers. Interestingly, disruption of PCIF1 methyltransferase activity slightly reversed the effect of PCIF1 overexpression on cell proliferation, but had no significant reversal effects on cell cycle progression or apoptosis. Knockdown of PCIF1 promoted the growth of gliomas, while overexpressing PCIF1 inhibited tumor growth and prolonged the survival time of tumor-bearing mice. In addition, the mRNA and protein levels of PCIF1 were gradually decreased with the increase of WHO grade in glioma tissues, but there was no significant correlation with patient survival. INTERPRETATION: These results indicated that PCIF1 played a suppressing role in glioma growth and survival, which may not entirely depend on its methyltransferase activity.

A novel biphenyl diester derivative, AB38b, inhibits glioblastoma cell growth via the ROS-AKT/mTOR pathway.

AB38b is a novel biphenyl diester derivative synthesized in our laboratory, and it has been shown to improve the pathology of nephropathy and encephalopathy in diabetic mice. Glioblastoma (GBM) is the most lethal brain tumor, without effective drugs to date. The present study aims at investigating the role of AB38b in GBM growth and revealing the underlying molecular mechanisms. We found that AB38b administration showed a dose- and time-dependent inhibition on cell proliferation in multiple immortalized and primary GBM cell lines, but it had no significant effects on human astrocyte cell line. More importantly, AB38b blocked cell cycle progression, induced early apoptosis, decreased the activity of AKT/mTOR pathway, and increased the generation of reactive oxygen species (ROS) in GBM cells. Interestingly, antioxidant treatments could reverse the AB38b-mediated abovementioned effects; overexpression of constitutively active AKT could partially rescue the suppressive effects of Ab38b on GBM cell proliferation. In addition, AB38b administration inhibited the tumor growth, decreased the activity of AKT/mTOR pathway, and prolonged the survival time in GBM animal models, without any adverse influences on the important organs. These findings suggest that AB38b exerts anti-glioma activity via elevating the ROS generation followed by inhibiting the activity of AKT/mTOR pathway.

Imipramine impedes glioma progression by inhibiting YAP as a Hippo pathway independent manner and synergizes with temozolomide.

Patients with malignant glioma often suffered from depression, which leads to an increased risk of detrimental outcomes. Imipramine, an FDA-approved tricyclic antidepressant, has been commonly used to relieve depressive symptoms in the clinic. Recently, imipramine has been reported to participate in the suppression of tumour progression in several human cancers, including prostate cancer, colon cancer and lymphomas. However, the effect of imipramine on malignant glioma is largely unclear. Here, we show that imipramine significantly retarded proliferation of immortalized and primary glioma cells. Mechanistically, imipramine suppressed tumour proliferation by inhibiting yes-associated protein (YAP), a recognized oncogene in glioma, independent of Hippo pathway. In addition to inhibiting YAP transcription, imipramine also promoted the subcellular translocation of YAP from nucleus into cytoplasm. Consistently, imipramine administration significantly reduced orthotopic tumour progression and prolonged survival of tumour-bearing mice. Moreover, exogenous overexpression of YAP partially restored the inhibitory effect of imipramine on glioma progression. Most importantly, compared with imipramine or temozolomide (TMZ) monotherapy, combination therapy with imipramine and TMZ exhibited enhanced inhibitory effect on glioma growth both in vitro and in vivo, suggesting the synergism of both agents. In conclusion, we found that tricyclic antidepressant imipramine impedes glioma progression by inhibiting YAP. In addition, combination therapy with imipramine and TMZ may potentially serve as promising anti-glioma regimens, thus predicting a broad prospect of clinical application.

BYSL Promotes Glioblastoma Cell Migration, Invasion, and Mesenchymal Transition Through the GSK-3ß/ß-Catenin Signaling Pathway.

BYSL, which encodes the human bystin protein, is a sensitive marker for astrocyte proliferation during brain damage and inflammation. Previous studies have revealed that BYSL has important roles in embryo implantation and prostate cancer infiltration. However, the role and mechanism of BYSL in glioblastoma (GBM) cell migration and invasion remain unknown. We found that knockdown of BYSL inhibited cell migration and invasion, downregulated the expression of mesenchymal markers (e.g., ß-catenin and N-cadherin), and upregulated the expression of epithelial marker E-cadherin in GBM cell lines. Overexpression of BYSL promoted GBM cell migration, invasion, and epithelial-mesenchymal transition (EMT). In addition, the role of BYSL in promoting EMT was further confirmed in a glioma stem cell line derived from a GBM patient. Mechanistically, overexpression of BYSL increased the phosphorylation of GSK-3ß and the nuclear distribution of ß-catenin. Inhibition of GSK-3ß by 1-Azakenpaullone could partially reverse the effects of BYSL downregulation on the transcriptional activity of ß-catenin, the expression of EMT markers, and GBM cell migration/invasion. Moreover, immunohistochemical analysis showed strong expression of BYSL in GBM tissues, which was positively correlated with markers of mesenchymal GBM. These results suggest that BYSL promotes GBM cell migration, invasion, and EMT through the GSK-3ß/ß-catenin signaling pathway.

PHAP1 promotes glioma cell proliferation by regulating the Akt/p27/stathmin pathway.

PHAP1 (Putative HLA-DR-associated protein 1), also termed acidic leucine-rich nuclear phosphoprotein 32A (ANP32A), Phosphoprotein 32 (pp32) or protein phosphatase 2A inhibitor (I1PP2A), is a multifunctional protein aberrantly expressed in multiple types of human cancers. However, its expression pattern and clinical relevance in human glioma remain unknown. In this study, Western blotting and immunohistochemistry analysis demonstrated PHAP1 protein was highly expressed in glioma patients, especially in those with high-grade disease. Publicly available data also revealed high levels of PHAP1 were associated with poor prognosis in glioma patients. The functional studies showed that knock-down of PHAP1 suppressed the proliferation of glioma cells, while overexpression of PHAP1 facilitated it. The iTRAQ proteomic analysis suggested that stathmin might be a potential downstream target of PHAP1. Consistently, PHAP1 knock-down significantly decreased the expression of stathmin, while overexpression of PHAP1 increased it. Also, the upstream negative regulator, p27, expression levels increased upon PHAP1 knock-down and decreased when PHAP1 was overexpressed. As a result, the phosphorylated Akt (S473), an upstream regulator of p27, expression levels decreased upon silencing of PHAP1, but elevated after PHAP1 overexpression. Importantly, we demonstrate the p27 down-regulation, stathmin up-regulation and cell proliferation acceleration induced by PHAP1 overexpression were dependent on Akt activation. In conclusion, the above results suggest that PHAP1 expression is elevated in glioma patients, which may accelerate the proliferation of glioma cells by regulating the Akt/p27/stathmin pathway.

The atypical protein kinase RIOK3 contributes to glioma cell proliferation/survival, migration/invasion and the AKT/mTOR signaling pathway.

The RIO (right open reading frame) protein kinases include RIOK1, RIOK2 and RIOK3. Emerging evidence has suggested an important role of RIO kinases in cancer cell proliferation, apoptosis, migration and invasion. However, the expression profile and specific roles of RIOK3 are largely unknown during glioma progression. In the current study, quantitative real-time PCR, Western blot, and immunohistochemical analysis showed that RIOK3 was upregulated in glioma tissues. Available database analysis revealed that higher levels of RIOK3 were associated with poorer survival outcome in glioma patients. Flow cytometry, CCK8 and EdU assays showed that downregulation of RIOK3 arrested cell cycle progression and inhibited glioma cell proliferation. Wound healing, transwell and gelatin zymography assays revealed that silencing RIOK3 decreased glioma cell migration and invasion. Furthermore, the downregulation of RIOK3 significantly decreased the activity of AKT/mTOR signaling and induced apoptosis in glioma cells. Overexpression of RIOK3 showed the opposite effects on glioma cell proliferation, migration, invasion and the AKT/mTOR pathway. These results indicate that high RIOK3 levels in gliomas appear to contribute to the growth and expansion of this cancer, and may thus serve as a novel therapeutic target.

Low Expression of CAPON in Glioma Contributes to Cell Proliferation via the Akt Signaling Pathway.

CAPON is an adapter protein for nitric oxide synthase 1 (NOS1). CAPON has two isoforms in the human brain: CAPON-L (long form of CAPON) and CAPON-S (short form of CAPON). Recent studies have indicated the involvement of CAPON in tumorigenesis beyond its classical role in NOS1 activity regulation. In this study, we found that the protein levels of CAPON-S, but not than CAPON-L, were significantly decreased in glioma tissues. Therefore, we established lentivirus-mediated stable cell lines with CAPON-S overexpression or down-regulation, and investigated the role of CAPON-S in the proliferation of glioma cells by using CCK8, EdU, and flow cytometry assays. Overexpression of CAPON-S reduced the cell variability and the percentage of EdU-positive cells, and arrested the cells in the G1 phase in glioma cells. Silencing of CAPON by short-hairpin RNA showed the opposite effects. Furthermore, an intracellular signaling array revealed that overexpression of CAPON-S resulted in a remarkable reduction in the phosphorylation of Akt and S6 ribosomal protein in glioma cells, which was further confirmed by Western blot. These findings suggest that CAPON may function as a tumor suppressor in human brain glioma and that the inactivation of the Akt signaling pathway caused by CAPON-S overexpression may provide insight into the underlying mechanism of CAPON in glioma cell proliferation.

Β-catenin is involved in Bex2 down-regulation induced glioma cell invasion/migration inhibition.

Previously, we found that brain expressed X-linked gene 2 (Bex2) regulates the invasion/migration ability of glioma cells. However, the mechanism of this effect remains unknown. In current study, we reported that Bex2 down-regulation inhibited glioma cell migration and invasion by decreasing the nucleus and cytoplasm protein level of ß-catenin. We found that the protein levels of Bex2 and ß-catenin were up-regulated and showed direct correlation in glioma tissues. Bex2 down-regulation significantly decreased ß-catenin protein levels but not its mRNA levels. Furthermore, the decreased protein level of ß-catenin was located in the nucleus and cytoplasm but not in the cell membrane. Further study found that the effects of Bex2 down-regulation on the invasion and migration of glioma cell could be reversed by ß-catenin over-expression. Taken together, Bex2 affects the invasion and migration ability of glioma cells by regulating ß-catenin.

FRK suppresses the proliferation of human glioma cells by inhibiting cyclin D1 nuclear accumulation.

The Fyn related kinase (FRK) is a noteworthy member of the Src non-receptor tyrosine kinase family for its distinctive tumor suppressive function. Recently, we have shown that FRK plays a protective role against the progression of glioma by suppressing cell migration and invasion. However, it is unclear whether the cell growth of glioma is also regulated by FRK and by which mechanism FRK alters its specific biological functions. In the current study, we found that FRK over-expression significantly suppressed the proliferation of glioma cells. In contrast, FRK knockdown by siRNA promoted glioma cell growth. In addition, FRK over-expression caused G1 phase arrest as well as apoptosis of glioma cells. Further investigation disclosed that FRK-induced G1 arrest was accompanied by down-regulation of hyperphosphorylated retinoblastoma protein (pRb), which led to the consequent suppression of E2F1. More importantly, we found that over-expression of FRK inhibited proper cyclin D1 accumulation in the nucleus of proliferating cells. Taken together, our results demonstrate a combined mechanism for the anti-proliferative effects of FRK by inhibiting cyclin D1 nucleus accumulation and pRb phosphorylation in glioma cells.

Bex2 controls proliferation of human glioblastoma cells through NF-κB signaling pathway.

Glioblastoma is the most common and fatal human brain malignancy in adults with highly proliferative capacity. Despite advances in surgery and adjuvant therapy, the median survival of patients has changed little over recent decades. Identifying molecules critical for glioma development is significant for devising effective targeted therapy. We previously reported that Bex2, a member of the brain expressed X-linked gene family, promoted the progression of glioma by promoting cell proliferation. In the present study, we investigated the main mechanism of Bex2 promoting the proliferation of glioblastoma cells. We found that Bex2 downregulation inhibited glioma cell proliferation and the expression of NF-κB p65, but Bex2 overexpression promoted them. Similarly, the proliferation of glioma cells was inhibited by p65 downregulation but increased by p65 overexpression. In addition, Bex2 overexpression-induced cell proliferation was abolished by p65 downregulation. Furthermore, Bex2 with nuclear localization signal deleted no longer promoted p65 expression. In conclusion, this study demonstrates that Bex2 promotes proliferation of human glioblastoma cells via NF-κB signaling pathway and Bex2 nuclear location is critical for p65 expression.

No association between polymorphisms in the calcium homeostasis modulator 1 gene and mesial temporal lobe epilepsy risk in a Chinese population.

PURPOSE: Mesial temporal lobe epilepsy (MTLE) is one of the most common forms of epilepsies in adults. The calcium homeostasis modulator 1 gene (CALHM1) has been considered one of the candidate genes that play a role in epileptogenesis due to its function in calcium homeostasis and amyloid ß (Aß) regulation. Recently, the association of a single nucleotide polymorphism (rs11191692) of CALHM1 has been reported to be associated with MTLE in Han Chinese, but independent replication is needed. In the present study, rs11191692 and rs2986017 of CALHM1 were determined in 512 MTLE patients and 412 control subjects to investigate the possible involvement of CALHM1 in the etiology of MTLE. METHOD: Genotyping was determined by polymerase chain reaction-restriction fragment length polymorphism method. Major statistical analyses were performed by SAS. RESULTS: No significant differences in the genotypic or allelic frequencies of both single-nucleotide polymorphisms were revealed between subjects with and without MTLE (rs11191692: P=0.890 and 0.230; rs2986017: P=0.581 and 0.072). Further stratification analysis by gender and age, and analysis of clinical features in relation to MTLE also yielded negative results. CONCLUSION: rs11191692 and rs2986017 of CALHM1 do not contribute substantially to MTLE in Han Chinese.

Bex2 is critical for migration and invasion in malignant glioma cells.

Glioblastoma is a kind of highly aggressive and incurable tumor with an average survival time of 12 months in our currently available treatment. The invasive cells are the sources of tumor recurrence and mechanisms of invasion are largely unknown. Identification of candidate genes important for invasion and migration is a hot spot of cancer biology. As one member of Bex protein family, Bex2 has its functions in the development of the nervous system and neurological diseases. Bex2 plays great roles in breast cancer, but its function and mechanisms in glioma progression remain unclear. In this study, we found Bex2 overexpression promoted cell migration and invasion, while Bex2 downregulation inhibited them. Meanwhile, we observed that Bex2 downregulation increased N-cadherin but decreased the excretion of MMP-2. Taken together, these data suggested that Bex2 promoted the progression of glioma by promoting cell migration and invasion, and these effects might be mediated by N-cadherin and MMP-2.

Bex2 regulates cell proliferation and apoptosis in malignant glioma cells via the c-Jun NH2-terminal kinase pathway.

The function of Bex2, a member of the Brain Expressed X-linked gene family, in glioma is controversial and its mechanism is largely unknown. We report here that Bex2 regulates cell proliferation and apoptosis in malignant glioma cells via the c-Jun NH2-terminal kinase (JNK) pathway. The expression level of Bex2 is markedly increased in glioma tissues. We observed that Bex2 over-expression promotes cell proliferation, while down-regulation of Bex2 inhibits cell growth. Furthermore, Bex2 down-regulation promotes cell apoptosis and activates the JNK pathway; these effects were abolished by administration of the JNK specific inhibitor, SP600125. Thus, Bex2 may be an important player during the development of glioma.