Search

Background Intensive chemotherapeutic regimens with craniospinal irradiation have greatly improved survival in medulloblastoma patients. However, survival markedly differs among molecular subgroups and their biomarkers are unknown. Through unbiased screening, we found Schlafen family member 11 (SLFN11), which is known to improve response to DNA damaging agents in various cancers, to be one of the top prognostic markers in medulloblastomas. Hence, we explored the expression and functions of SLFN11 in medulloblastoma. Methods SLFN11 expression for each subgroup was assessed by immunohistochemistry in 98 medulloblastoma patient samples and by analyzing transcriptomic databases. We genetically or epigenetically modulated SLFN11 expression in medulloblastoma cell lines and determined cytotoxic response to the DNA damaging agents cisplatin and topoisomerase I inhibitor SN-38 in vitro and in vivo. Results High SLFN11 expressing cases exhibited significantly longer survival than low expressing cases. SLFN11 was highly expressed in the WNT-activated subgroup and in a proportion of the SHH-activated subgroup. While WNT activation was not a direct cause of the high expression of SLFN11, a specific hypomethylation locus on the SLFN11 promoter was significantly correlated with high SLFN11 expression. Overexpression or deletion of SLFN11 made medulloblastoma cells sensitive and resistant to cisplatin and SN-38, respectively. Pharmacological upregulation of SLFN11 by the brain-penetrant histone deacetylase-inhibitor RG2833 markedly increased sensitivity to cisplatin and SN-38 in SLFN11-negative medulloblastoma cells. Intracranial xenograft studies also showed marked sensitivity to cisplatin by SLFN11-overexpression in medulloblastoma cells. Conclusions High SLFN11 expression is one factor which renders favorable outcomes in WNT-activated and a subset of SHH-activated medulloblastoma possibly through enhancing response to cisplatin.

Glioma-associated oncogene homolog 3 (GLI3), whose main function is to inhibit GLI1, has been associated with neuronal differentiation in medulloblastoma. However, it is not clear what molecular subtype(s) show increased GLI3 expression. GLI3 levels were assessed by immunohistochemistry in 2 independent cohorts, including a total of 88 cases, and found to be high in both WNT- and SHH-activated medulloblastoma. Analysis of bulk mRNA expression data and single cell RNA sequencing studies confirmed that GLI1 and GLI3 are highly expressed in SHH-activated medulloblastoma, whereas GLI3 but not GLI1 is highly expressed in WNT-activated medulloblastoma. Immunohistochemical analysis has shown that GLI3 is expressed inside the neuronal differentiated nodules of SHH-activated medulloblastoma, whereas GLI1/2 are expressed in desmoplastic areas. In contrast, GLI3 is diffusely expressed in WNT-activated medulloblastoma, whereas GLI1 is suppressed. Our data suggest that GLI3 may be a master regulator of neuronal differentiation and morphology in these subgroups.

BACKGROUND: We have previously investigated the expression of Gli3, a downstream target of the Sonic Hedgehog pathway, which main function is to suppress Gli1/2 in medulloblastomas. We found that Gli3 is associated with neuronal and glial differentiation in desmoplastic / nodular (D/N) type medulloblastomas (Miyahara et al., Neuropathology, 2013). In the present study, we investigated the expression of Gli3 in the molecular subgroups. METHOD: Thirty-one medulloblastomas treated at Department of Neurosurgery, Niigata University between 1982 and 2013 were retrospectively studied. Molecular classification into 4 subgroups (WNT-activated, SHH-activated, Group 3 and Group 4) was performed using Nanostring. HE and NeuN, GFAP, beta-catenin, GAB-1, and YAP-1 immunohistochemistry was performed. Furthermore, Gli3 and Gli1 expression in each molecular subgroup was assessed in the public data base R2. RESULTS: Nanostring was considered reliable (confidence > 0.9) in 28 cases. Four cases were classified as WNT- activated, 5 cases as SHH-activated, 4 cases as Group 3 and 16 cases as Group 4. Gli3 was positive in 7 out of 9 (78%) WNT-/SHH- cases, whereas Gli3 was positive in only 8 out of 19 (42.1%) non-WNT-/SHH- subgroup (p = 0.1145, Fishers exact test). R2 database analysis confirmed that Gli3 was significantly elevated in WNT- and SHH-activated medulloblastoma. Gli1 was elevated in SHH-activated cases but suppressed in WNT-activated cases. IHC analysis revealed that Gli3 was elevated inside the nodules of D/N type medulloblastoma. Neuronal differentiation was seen in these nodules. CONCLUSION: These results suggest that Gli3 is elevated inside the nodules of SHH-activated medulloblastoma, whereas in WNT-activated cases, Gli3 suppresses HH signaling in the entire tumor.

BACKGROUND: Diffuse intrinsic pontine glioma (DIPG) is an invasive and treatment-refractory pediatric brain tumor. The Target of Rapamycin Complex 1 and 2 (mTORC1/2) regulate protein translation, cell growth, survival, invasion and metabolism. Primary DIPG tumors harbor mutations in PTEN, AKT, and PI3K. Inhibition of TORC1 with rapalogs is minimally effective in DIPG, however, the efficacy of dual mTOR kinase inhibitors has not been examined in this tumor type. METHODS: Three DIPG cell lines (JHHDIPG1, SF7761, and SUDIPG XIII), maintained in medium with EGF and FGF, were treated for 4-72 hours with DMSO or 1-50 nM levels of the TORC1/2 kinase inhibitor MLN0128. Cell growth analysis was performed using MTS reagent and measured in 490-nm absorbance. Protein was extracted from cell lysates and Western blotting was performed using primary antibodies specific for pAKT, AKT, pS6, S6, and cleaved PARP, as well as β-actin. Immunofluorescence staining against cleaved caspase 3 (CC3) and BrdU were done to evaluate apoptosis and proliferation, respectively. RESULTS: Both pAKT and pS6 were highly expressed in these DIPGs cell lines. pAKT and pS6 were inhibited after 25nM and 10-25nM levels of MLN0128, respectively. Cell growth of DIPGs (MTS assay) was markedly inhibited by 25-50 nM MLN0128 (SF7761: 10 nM p = 0.0002, 25/50 nM p < 0.0001; SUDIPG XIII: 25 nM p = 0.0065, 50 nM p = 0.0003, all vs vehicle). Decreased proliferation (%BrdU: JHHDIPG1 p = 0.0001, SF7761 p < 0.0001, SUDIPG XIII p < 0.0003, all vehicle vs 25 nM) and increased apoptosis (%CC3: JHHDIPG1 p = 0.0004, SF7761 p = 0.0005, SUDIPG XIII p = 0.0032, all vehicle vs 25 nM; increased cleaved PARP) were seen after treatment. CONCLUSIONS: In these DIPG cell lines, the mTOR pathway is active, and inhibition of the pathway suppresses growth and induces apoptosis. These effects may be induced as a consequence of inhibition of both mTORC1 and mTORC2. Dual mTOR inhibition is a promising candidate for DIPG treatment.