Kay Ka Wai Li

Minichromosome maintenance proteins 2, 3 and 7 in medulloblastoma: overexpression and involvement in regulation of cell migration and invasion

Minichromosome maintenance (MCM) proteins 2–7 are important in DNA replication licensing. Functional roles beyond licensing are speculated. In addition, significances in medulloblastoma (MB) remain unclear. In this study, we showed the frequent deregulation of MCM2 and MCM3 expression in 7 MB cell lines and 31 clinical samples. Moreover, DAOY and ONS76 and the clinical samples expressed elevated MCM7 transcripts with genomic gain of the gene. Immunopositivity restricted to tumor cells was found in 41, 37 and 53 out of 73 MB cases for MCM2, MCM3 and MCM7, respectively. High-MCM3 expression was associated with poor prognosis. Knockdowns of these MCMs significantly inhibited anchorage-dependent and -independent MB cell growth. The inhibition of MCM3 expression by small interfering RNA knockdown was related to G1 arrest with reduced cyclin A expression, whereas the MCM2- and MCM7-knocked-down cells arrested at G2/M with increased cyclin A expression. Interestingly, we demonstrated the links of these MCMs with cell migration and invasion using wound-healing and Transwell migration/invasion assays. Exogenous overexpression of MCM2, MCM3 and MCM7 increased anchorage-independent cell growth, and also cell migration and invasion capabilities in MB cells. The knockdown reduced the number of filopodial cells and the cells with intense stress fibers by blocking cdc42 and Rho activation. Taken together, deregulation of MCM2, MCM3 and MCM7 expression might be involved in MB tumorigenesis and we revealed undefined roles of these MCMs in control of MB cell migration and invasion.

MiR-383 is Downregulated in Medulloblastoma and Targets Peroxiredoxin 3 (PRDX3)

Accumulating evidence suggests that microRNAs (miRNAs) are over‐ or under‐expressed in tumors, and abnormalities in miRNA expression may contribute to carcinogenesis. MiR‐383 was previously identified as one of the under‐expresssed miRNAs in medulloblastoma (MB) by miRNA expression profiling. Quantitative reverse transcription polymerase chain reaction (RT‐PCR)‐based miRNA assays showed an enrichment of miR‐383 in normal brain. Based on these data, we speculated that miR‐383 is important in MB pathogenesis. In this study, we demonstrated significant downregulation of miR‐383 in 23/29 (79%) MB samples and 7/7 (100%) MB cells lines. Ectopic expression of miR‐383 in MB cells led to suppression of cell growth, cell accumulation at sub‐G1 phase and alteration of apoptosis‐related proteins. By transcriptomic analysis and computational algorithms, we identified peroxiredoxin 3 (PRDX3) as a target gene of miR‐383. Luciferase reporter assay confirmed that miR‐383 negatively regulated PRDX3 by interaction between miR‐383 and complementary sequences in the 3′ UTR of PRDX3. MiR‐383 repressed PRDX3 at transcriptional and translational levels as revealed by quantitative RT‐PCR and Western blot analysis. Furthermore, depletion of PRDX3 by siRNAs resulted in similar effects as observed in miR‐383‐transfected cells. In conclusion, miR‐383 acts as a regulator controlling cell growth of MB, at least in part, through targeting PRDX3.

TERT promoter mutations contribute to subset prognostication of lower-grade gliomas

Recurrent mutations in the promoter region of telomerase reverse transcriptase (TERT) have been found in various cancers including diffuse gliomas. Mutations lead to TERT upregulation and are associated with aggressive clinical behavior in glioblastomas. However, the clinical significance of TERT promoter mutations in lower-grade gliomas remains undetermined. The aim of this study is to evaluate the status of TERT promoter and the respective prognostic significance in a cohort of 237 lower-grade gliomas comprising grades II and III astrocytomas, oligodendrogliomas, and oligoastrocytomas. Mutually exclusive mutations in TERT promoter, C228T and C250T, were identified in 16/105 (15%) diffuse astrocytomas, 16/63 (25%) anaplastic astrocytomas, 13/18 (72%) oligodendrogliomas, 3/3 (100%) anaplastic oligodendrogliomas, 17/45 (38%) oligoastrocytomas, and 2/3 (67%) anaplastic oligoastrocytomas. Mutations co-occurred with 1p/19q codeletion (P<0.001) and are associated with oligodendroglial histology (P<0.001). Kaplan–Meier’s survival analysis showed that TERT promoter mutation (P=0.037), Isocitrate dehydrogenase (IDH) mutation (P<0.001), and 1p/19q codeletion (P<0.001) were associated with favorable overall survival (OS). In the subset of 116 IDH-mutated lower-grade gliomas lacking 1p/19q codeletion, 19 TERT promoter-mutated tumors exhibited longer progression-free survival (PFS) (P=0.027) and OS (P=0.004). Consistent with this observation, in the subset of 97 IDH-mutated astrocytomas, 14 TERT promoter-mutated tumors showed longer PFS (P=0.001) and OS (P=0.001). In contrast, among the subset of 74 IDH wild-type lower-grade gliomas with intact 1p/19q, TERT promoter mutation was associated with shorter PFS (P=0.001) and OS (P=0.001). Similarly, in the subset of 65 IDH wild-type astrocytomas, 16 TERT promoter-mutated tumors exhibited unfavorable PFS (P=0.007) and OS (P=0.008). Our results indicate that when combined with IDH status, TERT promoter mutation contributes to prognostic subgroups of lower-grade astrocytic tumors or 1p/19q intact lower-grade gliomas and this may further refine future molecular classification of lower-grade gliomas.

miR-106b is overexpressed in medulloblastomas and interacts directly with PTEN

MicroRNAs (miRNAs) are an abundant group of small non‐coding RNAs that have been implicated in tumorigenesis. They regulate expression of target genes by complementary base pairing. The purposes of this study were to delineate miR‐106b expression in medulloblastoma (MB) and to explore its functional contributions to MB pathogenesis.

Loss of CIC and FUBP1 expressions are potential markers of shorter time to recurrence in oligodendroglial tumors

Combined deletion of chromosomes 1p and 19q is a prognostic marker in oligodendroglial tumors. Recent studies in oligodendroglial tumors have unveiled recurrent mutations of CIC (homolog of Drosophila capicua) and FUBP1 (far upstream element binding protein 1) that are located on 19q13 and 1p31, respectively. However, the impact of CIC and FUBP1 mutations on their protein expressions has not been examined. The aims of this study were to correlate the expression patterns of CIC and FUBP1 with their mutation profiles and to evaluate the clinical relevance of these molecular markers in 55 oligodendroglial tumors diagnosed in 47 adult patients. Using direct sequencing, somatic mutations of CIC and FUBP1 were identified in 47% (22/47) and 16% (7/45) of oligodendroglial tumors, respectively. Immunohistochemical analysis revealed loss of CIC or FUBP1 protein expression in 36% (20/55) and 16% (9/55) of oligodendroglial tumors examined. Somatic mutation was significantly associated with absent protein expression for both genes (CIC, P=0.01; FUBP1, P=0.00001). Four tumors with undetectable CIC mutations exhibited absent CIC expression, suggesting that CIC inactivation could be mediated by mechanisms other than mutations and genomic loss. Univariate survival analysis revealed that 1p/19q codeletion was significantly associated with overall survival (P=0.05). Loss of CIC expression was significantly correlated with shorter progression-free survival (P=0.03), whereas CIC alteration (mutation or null expression) with worse overall survival (P=0.05). Absent FUBP1 expression was linked with unfavorable progression-free survival (P=0.02) and overall survival (P=0.01). In 16 tumors with 1p/19q codeletion, CIC mutation was associated with unfavorable survival (P=0.01). There was a correlation between lack of CIC or FUBP1 expression and poor progression-free survival (P=0.004; P=0.0003). No molecular markers showed association with survival in oligodendroglial tumors lacking 1p/19q codeletion. We conclude that absent CIC and FUBP1 expressions are potential markers of shorter time to recurrence and CIC mutation a potential marker of worse prognosis, especially in tumors carrying 1p/19q codeletion.

MIR‐137 Suppresses Growth and Invasion, is Downregulated in Oligodendroglial Tumors and Targets CSE1L

MicroRNA‐137 (miR‐137) expression has been reported to be decreased in astrocytic tumors in two expression profiling studies but its role in the pathogenesis of oligodendroglial tumors is still limited. In this study, we demonstrate that miR‐137 expression is significantly downregulated in a cohort of 35 oligodendroglial tumors and nine glioma cell lines compared with normal brains. Lower miR‐137 expression is associated with shorter progressive‐free survival and overall survival. Restoration of miR‐137 expression in an oligodendroglial cells TC620, and also glioblastoma cells of U87 and U373 significantly suppressed cell growth, anchorage‐independent growth, as well as invasion. Demethylation and deacetylation treatments resulted in upregulation of miR‐137 expression in TC620 cells. In silico analysis showed that CSE1 chromosome segregation 1‐like (yeast) (CSE1L) is a potential target gene of miR‐137. Luciferase reporter assay demonstrated that miR‐137 negatively regulates CSE1L by interaction between miR‐137 and complementary sequences in the 3′ UTR of CSE1L. Immunohistochemistry revealed that CSE1L is upregulated in oligodendroglial tumors. Knockdown of CSE1L resulted in similar outcomes as overexpressing miR‐137 in oligodendroglioma cells and glioblastoma cells. Overall, our data suggest that miR‐137 regulates growth of glioma cells and targets CSE1L, providing further understanding in the tumorigenesis of gliomas.

Biomarker-based prognostic stratification of young adult glioblastoma

While the predominant elderly and the pediatric glioblastomas have been extensively investigated, young adult glioblastomas were understudied. In this study, we sought to stratify young adult glioblastomas by BRAF, H3F3A and IDH1 mutations and examine the clinical relevance of the biomarkers. In 107 glioblastomas aged from 17 to 35 years, mutually exclusive BRAF-V600E (15%), H3F3A-K27M (15.9%), H3F3A-G34R/V (2.8%) and IDH1-R132H (16.8%) mutations were identified in over half of the cases. EGFR amplification and TERTp mutation were only detected in 3.7% and 8.4% in young adult glioblastomas, respectively. BRAF-V600E identified a clinically favorable subset of glioblastomas with younger age, frequent CDKN2A homozygous deletion, and was more amendable to surgical resection. H3F3A-K27M mutated glioblastomas were tightly associated with midline locations and showed dismal prognosis. IDH1-R132H was associated with older age and favorable outcome. Interestingly, tumors with positive PDGFRA immunohistochemical expression exhibited poorer prognosis and identified an aggressive subset of tumors among K27M mutated glioblastomas. Combining BRAF, H3F3A and IDH1 mutations allowed stratification of young adult glioblastomas into four prognostic subgroups. In summary, our study demonstrates the clinical values of stratifying young adult glioblastomas with BRAF, H3F3A and IDH1 mutations, which has important implications in refining prognostic classification of glioblastomas.

KIAA0495/PDAM Is frequently downregulated in oligodendroglial tumors and its knockdown by siRNA induces cisplatin resistance in glioma cells

Co‐deletion of chromosomes 1p and 19q is a common event in oligodendroglial tumors (OTs), suggesting the presence of OT‐related genes. The aim of this study was to identify the target genes residing in the minimally deleted regions on chromosome 1p36.31–p36.32 that might be involved in OTs. A novel gene KIAA0495/p53‐dependent apoptosis modulator (PDAM) was found frequently deregulated, with 37 of 58 (63.8%) OTs examined showing reduced expression compared with normal brain. Chromosome 1p loss and epigenetic modifications were the major mechanisms contributing to PDAM downregulation. The role of PDAM in chemosensitivity was also evaluated. PDAM knockdown had no effect on sensitivity to vincristine, lomustine, temozolomide and paclitaxel, but could induce cisplatin resistance in glioma cells harboring wild‐type p53. B‐cell CCL/lymphoma 2 (BCL2)‐like 1 (BCL2L1) exhibited significant upregulation, while BCL2 showed partial derepression in PDAM‐silenced cells after cisplatin treatment, suggesting that alteration of anti‐apoptotic genes contributed in part to cisplatin resistance. Knockdown of BCL2L1 abrogated the induced cisplatin‐resistant phenotype. Moreover, our data suggested that PDAM might function as a non‐protein‐coding RNA. Collectively, these findings suggest that PDAM deregulation may play a role in OT development and that PDAM may possess the capacity to modulate apoptosis via regulation of p53‐dependent anti‐apoptotic genes.

EMP3 overexpression is associated with oligodendroglial tumors retaining chromosome arms 1p and 19q

The epithelial membrane protein 3 (EMP3) gene located on chromosome 19q13 has been implicated as a candidate tumor suppressor gene (TSG) in neuroblastomas and gliomas. The aim of this study was to investigate whether EMP3 is involved in oligodendroglial tumors (OTs), which frequently carry combined chromosomes 1p and 19q deletion. We first investigated the transcript level of EMP3 in a cohort of 57 OTs by quantitative real‐time RT‐PCR. Our results showed that 10 (18%) tumors had reduced EMP3 expression level compared to normal brains. Six of these tumors carried chromosome 19q13 deletion but no statistical correlation was found between the 2 parameters. Intriguingly, a similar proportion (11 of 57, 19%) of tumors displayed EMP3 overexpression, with 8 of them having transcript level >10‐fold higher than normal brain. All 11 OTs retained chromosomes 1p36 and 19q13, and a significant association was found between EMP3 overexpression and balanced chromosomes 1p36 and 19q13 (p = 0.004). The methylation status of EMP3 was evaluated by bisulfite sequencing in 29 OTs with diverse expression levels. All tumors except 3 showed aberrant methylation of EMP3 and no correlation was observed between transcript level and methylation status, suggesting that methylation alone does not mediate transcriptional down‐regulation of EMP3 in OTs. In conclusion, our study demonstrates that EMP3 overexpression is involved in OTs retaining chromosomes 1p and 19q and does not support EMP3 as the target TSG on chromosome 19q13 in OTs.

Not all 1p/19q non-codeleted oligodendroglial tumors are astrocytic

Although 1p/19q codeletion is the genetic hallmark defining oligodendrogliomas, approximately 30-40% of oligodendroglial tumors have intact 1p/19q in the literature and they demonstrate a worse prognosis. This group of 1p/19q intact oligodendroglial tumors is frequently suggested to be astrocytic in nature with TP53 and ATRX mutations but actually remains under-investigated. In the present study, we provided evidence that not all 1p/19q intact oligodendroglial tumors are astrocytic through histologic and molecular approaches. We examined 1p/19q status by FISH in a large cohort of 337 oligodendroglial tumors and identified 39.8% lacking 1p/19q codeletion which was independently associated with poor prognosis. Among this 1p/19q intact oligodendroglial tumor cohort, 58 cases demonstrated classic oligodendroglial histology which showed older patient age, better prognosis, association with grade III histology, PDGFRA expression, TERTp mutation, as well as frequent IDH mutation. More than half of the 1p/19q intact oligodendroglial tumors showed lack of astrocytic defining markers, p53 expression and ATRX loss. TP53 mutational analysis was additionally conducted in 45 cases of the 1p/19q intact oligodendroglial tumors. Wild-type TP53 was detected in 71.1% of cases which was associated with classic oligodendroglial histology. Importantly, IDH and TERTp co-occurred in 75% of 1p/19q intact, TP53 wild-type oligodendrogliomas, highlighting the potential of the co-mutations in assisting diagnosis of oligodendrogliomas in tumors with clear cell morphology and non-codeleted 1p/19q status. In summary, our study demonstrated that not all 1p/19q intact oligodendroglial tumors are astrocytic and co-evaluation of IDH and TERTp mutation could potentially serve as an adjunct for diagnosing 1p/19q intact oligodendrogliomas.