Pomoter Methylation-Associated Loss of ID4 Expression Have a Prognostic Relevance in Glioblastoma Multiforme
Maurizio Martini, Tonia Cenci, Nicola Montano, Valeriana Cesarini, Roberto Pallini, Luigi M Larocca. Università Cattolica del Sacro Cuore, Rome, Italy
Background: Inhibitors of DNA binding/differentiation (ID1-4) are a family of helix-loop-helix transcription factor, highly expressed during embryogenesis and at lower levels in mature tissues. Expression of ID proteins have related to features of malignancies, including cellular transformation, metastasis and angiogenesis. ID4 plays an important role in neuronal stem cell differentiation and its deregulation has been implicated in glial neoplasia. Recent works have shown that hypermethylation of ID4 can determine the silencing of this protein in several human cancers.
Design: We analyzed the methylation status of ID4 by methylation-specific-PCR in 65 glioblastoma multiforme (GBM) cases and in 20 normal brain tissues. After surgical treatment, all GBMs were subjected to adjuvant radiotherapy with concomitant administration of temozolomide. Methylation status of ID4 was confirmed by sequencing after subcloning and RNA and protein expression was assessed by real-time PCR and immunohistochemistry. We also evaluated the mRNA expression of MGP (Matrix GLA protein). The relationship between ID4 methylation and clinical outcome was investigated.
Results: The promoter of ID4 was methylated in 17/65 (26%) GBMs. After subcloning, the methylation was confirmed by sequencing in over 95% of GpC dinucleotides. In methylated GBMs, ID4 mRNA expression was reduced 5 folds as compared with unmethylated GBMs. A significant reduction of protein expression was detected in all hypermethylated cases. Moreover, GBM with ID4 methylation showed a reduced MGP mRNA expression by 2 folds in comparison to GBM with unmethyalted ID4. ID4 methylation was significantly associated with a favourable clinical outcome (p=0.0075). No significant differences were found between patients with and without ID4 methylation with respect to age, sex, p53 mutation, Ki67 labeling index and extent of surgical resection.
Conclusions: Our data suggest that methylation of ID4 may be involved in the pathogenesis of GBM probably playing a role on MGP mediated neoangiogenesis, and in the resistance of this neoplasm to conventional treatment.
Tuesday, March 20, 2012 1:15 PM
Platform Session: Section F, Tuesday Afternoon