Downregulation of PRDX1 by promoter hypermethylation is frequent in 1p/19q-deleted oligodendroglial tumours and increases radio- and chemosensitivity of Hs683 glioma cells in vitro.

Deletions of chromosomal arms 1p and 19q are frequent in oligodendroglial tumours and linked to radio- and chemotherapy response as well as longer survival. The molecular mechanisms underlying this clinically important association are as yet unknown. Here, we studied the peroxiredoxin 1 (PRDX1) gene at 1p34.1 for promoter methylation and expression in primary gliomas and investigated its role in radio- and chemosensitivity of glioma cells in vitro. In total, we screened primary glioma tissues from 93 patients for methylation of the 5'-CpG island of PRDX1 by sodium bisulfite sequencing. PRDX1 mRNA and protein expression levels were determined in subsets of the tumours by quantitative PCR and western blot analysis, respectively. PRDX1 hypermethylation and reduced expression were frequently detected in oligodendroglial tumours and secondary glioblastomas, but not in primary glioblastomas. In oligodendroglial tumours, both PRDX1 hypermethylation and reduced mRNA expression were significantly associated with 1p/19q-deletion. Stable knockdown of PRDX1 by lentiviral transduction of short-hairpin (sh)RNA constructs significantly increased apoptosis and reduced cell viability of Hs683 glioma cells exposed to ionizing irradiation or temozolomide in vitro. Taken together, our findings indicate that epigenetic silencing of PRDX1 is frequent in 1p/19q-deleted oligodendroglial tumours and likely contributes to radio- and chemosensitivity of these tumours.

RNAi screening in glioma stem-like cells identifies PFKFB4 as a key molecule important for cancer cell survival.

The concept of cancer stem-like cells (CSCs) has gained considerable attention in various solid tumors including glioblastoma, the most common primary brain tumor. This sub-population of tumor cells has been intensively investigated and their role in therapy resistance as well as tumor recurrence has been demonstrated. In that respect, development of therapeutic strategies that target CSCs (and possibly also the tumor bulk) appears a promising approach in patients suffering from primary brain tumors. In the present study, we utilized RNA interference (RNAi) to screen the complete human kinome and phosphatome (682 and 180 targets, respectively) in order to identify genes and pathways relevant for the survival of brain CSCs and thereby potential therapeutical targets for glioblastoma. We report of 46 putative candidates including known survival-related kinases and phosphatases. Interestingly, a number of genes identified are involved in metabolism, especially glycolysis, such as PDK1 and PKM2 and, most prominently PFKFB4. In vitro studies confirmed an essential role of PFKFB4 in the maintenance of brain CSCs. Furthermore, high PFKFB4 expression was associated with shorter survival of primary glioblastoma patients. Our findings support the importance of the glycolytic pathway in the maintenance of malignant glioma cells and brain CSCs and imply tumor metabolism as a promising therapeutic target in glioblastoma.

Expression of an ASCL2 related stem cell signature and IGF2 in colorectal cancer liver metastases with 11p15.5 gain.

BACKGROUND AND AIMS: Liver metastases are the leading cause of death in colorectal cancer. To gain better insight into the biology of metastasis and possibly identify new therapeutic targets we systematically investigated liver-metastasis-specific molecular aberrations. METHODS: Primary colorectal cancer (pCRC) and matched liver metastases (LMs) from the same patients were analysed by microarray-based comparative genomic hybridisation in 21 pairs and gene expression profiling in 18 pairs. Publicly available databases were used to confirm findings in independent datasets. RESULTS: Chromosome aberration patterns and expression profiles of pCRC and matched LMs were strikingly similar. Unsupervised cluster analysis of genomic data showed that 20/21 pairs were more similar to each other than to any other analysed tumour. A median of only 11 aberrations per patient was found to be different between pCRC and LM, and expression of only 16 genes was overall changed upon metastasis. One region on chromosome band 11p15.5 showed a characteristic gain in LMs in 6/21 patients. This gain could be confirmed in an independent dataset of LMs (n=50). Localised within this region, the growth factor IGF2 (p=0.003) and the intestinal stem cell specific transcription factor ASCL2 (p=0.029) were found to be over-expressed in affected LM. Several ASCL2 target genes were upregulated in this subgroup of LM, including the intestinal stem cell marker OLFM4 (p=0.013). The correlation between ASCL2 expression and four known direct transcriptional targets (LGR5, EPHB3, ETS2 and SOX9) could be confirmed in an independent expression dataset (n=50). CONCLUSIONS: With unprecedented resolution a striking conservation of genomic alterations was demonstrated in liver metastases, suggesting that metastasis typically occurs after the pCRC has fully matured. In addition, we characterised a subset of liver metastases with an ASCL2-related stem-cell signature likely to affect metastatic behaviour of tumour cells.

Hypermethylation and transcriptional downregulation of the CITED4 gene at 1p34.2 in oligodendroglial tumours with allelic losses on 1p and 19q.

Deletions of chromosomal arms 1p and 19q are frequent in oligodendroglial tumours and have been associated with sensitivity to radio- and chemotherapy as well as favourable prognosis. By using microarray-based expression profiling, we found that oligodendroglial tumours with 1p and 19q losses showed significantly lower expression of the CBP/p300-interacting transactivator with glutamic acid/aspartic acid-rich carboxyl-terminal domain 4 gene (CITED4) at 1p34.2 as compared to tumours without 1p and 19q losses. Mutational analysis showed no CITED4 mutations in gliomas. However, 1p and 19q losses as well as low expression of CITED4 transcripts were significantly associated with hypermethylation of the CITED4-associated CpG island. In line with the latter finding, treatment of CITED4 hypermethylated glioma cell lines with 5-aza-2'-deoxycytidine and trichostatine A resulted in a marked increase of the CITED4 transcript levels. Furthermore, CITED4 hypermethylation was significantly associated with longer recurrence-free and overall survival of patients with oligodendroglial tumours. Taken together, our results indicate that CITED4 is epigenetically silenced in the vast majority of oligodendroglial tumours with 1p and 19q deletions and suggest CITED4 hypermethylation as a novel prognostic marker in oligodendroglioma patients.