Epigenetic repression of RASSF1A but not CASP8 in supratentorial PNET (sPNET) and atypical teratoid/rhabdoid tumors (AT/RT) of childhood

Abstract

Supratentorial primitive neuroectodermal tumors (sPNET) and atypical teratoid/rhabdoid tumors (AT/RT) of the CNS represent a biological and clinical enigma, despite advances in both molecular techniques and clinical management for these two rare embryonal brain tumors of childhood. Epigenetic changes hold great potential as possible disease mechanisms and may be manipulated therapeutically. We thus studied aberrant methylation of the genes RASSF1A and CASP8 and its consequence on expression in cell lines and primary tumors using a combination of semiquantitative methylation specific PCR (MSP), bisulfite sequencing and RT-PCR. In all, 17 samples of autopsy-derived normal appearing brain served as controls. Opposed to control tissues 19/24 sPNET and 4/6 AT/RT demonstrated aberrant methylation for the RASSF1A promoter region. Treatment of cell lines using 5-Aza-2 0 -deoxycytidine (5AZA) alone or in combination with trichostatin A (TSA) succeeded in re-establishing expression of RASSF1A in cell lines derived from a renal rhabdoid, an AT/RT and a medulloblastoma. A 5 0 CpG-rich region of CASP8 was methylated in normal tissues and in tumors. However, CASP8 showed inconsistent expression patterns in normal and tumor tissues. Our results indicate that aberrant methylation of the RASSF1A promoter region may be of importance in the origin and progression of sPNET and AT/ RT while the analysed 5 0 -CpG rich region of the CASP8 gene does not seem to play an important role in these tumors. Further studies of epigenetic changes in these rare tumors are warranted as their biology remains obscure and treatment efforts have been rather unsuccessfull.

Inhibitors of histone deacetylases as potential therapeutic tools for high‐risk embryonal tumors of the nervous system of childhood

“…Data from our laboratory show that aberrant methylation of the tumor suppressor gene RASSF1A can be found in 4 out of 6 AT/RT, and reexpression of the gene could be achieved by a combination of the demethylating agent 5-aza-2 0 -deoxycytidine and TSA. 20 In our study we find HDI highly effective towards the mentioned tumors, supporting the need for more preclinical studies especially concerning high-risk medulloblastoma, neuroblastoma and rhabdoid tumors. In a novel approach, we enclosed tumor cell lines from this entity in our investigation.…”

“…We used RT-PCR on genes, which had previously been found epigenetically silenced in cell lines. 20 Experimental procedures have been described. 20 Primer sequences were:…”

Epigenetic mechanisms regulating neural development and pediatric brain tumor formation

“…This genetic alteration appears to be crucial in the molecular pathogenesis of most AT/RTs. 122 Mühlisch et al 105 found the aberrant methylation of the RASSF1A promotor region as having an important role in AT/RT formation and progression. The same group evaluated the effects of HDAC inhibitors in embryonal tumor cell lines demonstrating a dose-and time-dependent apoptosis.…”

Quantitative analysis of DNA methylation in the promoter region of the methylguanine‐O6‐DNA‐methyltransferase gene by COBRA and subsequent native capillary gel electrophoresis

“…For this purpose, DNA clones representing methylated and non‐methylated bisulfite‐converted DNA at position C122 and C229 were amplified separately by PCR. Then the PCR products were mixed to obtain the theoretical methylation levels given above . These artificial samples were used to prove the key parameters of the developed analytical method, such as linearity, migration times, accuracy and precision of the recorded data sets (“proof‐of‐principle”).…”