Phosphatidylinositol 3′-Kinase/AKT Signaling Is Activated in Medulloblastoma Cell Proliferation and Is Associated with Reduced Expression of PTEN

Abstract

Purpose: Medulloblastomas represent the most frequent malignant brain tumors of childhood.They are supposed to originate from cerebellar neural precursor cells. Recently, it has been shown that Sonic Hedgehog^induced formation of medulloblastoma in an animal model is significantly enhanced by activation of the phosphatidylinositol 3 ¶-kinase (PI3K) signaling pathway. Experimental Design: To examine a role for PI3K/AKT signaling in the molecular pathogenesis of human medulloblastoma, we did an immunohistochemical study of the expression of Ser 473 -phosphorylated (p)-AKT protein in 22 medulloblastoma samples: All samples displayed p-AKT expression. To investigate if an activated PI3K/AKT pathway is required for medulloblastoma cell growth, we treated five human medulloblastoma cell lines with increasing concentrations of the PI3K inhibitor LY294002 and analyzed cellular proliferation and apoptosis. The antiproliferative effect could be antagonized by overexpressing constitutively active AKT. As the activation of PI3K/AKT signaling may be associated with alterations of the PTEN gene located at 10q23.3, a chromosomal region subject to frequent allelic losses in medulloblastoma, we screened PTEN for mutations and mRNA expression. Results: Proliferation of all of the medulloblastoma cell lines was dependent on PI3K/AKT signaling, whereas apoptosis was not prominently affected. Allelic loss was detected in 16% of the cases. One medulloblastoma cell line was found to carry a truncating mutation in the PTEN coding sequence. Even more important, PTEN mRNA and protein levels were found to be significantly lower in medulloblastomas compared with normal cerebellar tissue of different developmental stages. Reduction of PTEN expression was found to be associated with PTEN promoter hypermethylation in 50% of the tumor samples. Conclusions: We conclude that activation of the PI3K/AKT pathway constitutes an important step in the molecular pathogenesis of medulloblastoma and that dysregulation of PTEN may play a significant role in this context.

PTEN signaling in brain: neuropathology and tumorigenesis

“…Indeed, it is possible that AKT1, AKT3 or both isoforms may play crucial roles in brain tumorigenesis given their shared contribution to embryonic development of the CNS . Although elevation of AKT activity is seen more frequently than PTEN mutation in gliomas and medulloblastomas (Schlegel et al, 2002;Hartmann et al, 2006), mutations causing increased activity of any AKT isoform have not been observed frequently. AKT1 amplification has been reported in various human cancers, including a single case of gliosarcoma (Knobbe and Reifenberger, 2003) and AKT3 mutation has been reported in a single case of glioma (Hunter et al, 2006); however, the consequence of this missense mutation (G171R) on the activity of the enzyme is unknown.…”

“…Large scale analysis of genomic gains and loss from over 200 medulloblastoma cases found that loss of 10q is a frequent event (Rickert and Paulus, 2004). Despite this, only rare cases of PTEN mutation have been identified in these tumors (Rasheed et al, 1997), instead observing lower levels of PTEN mRNA expression, potentially caused by epigenetic modifications (Hartmann et al, 2006). An important role of PI3K pathway activation in medulloblastoma was recently suggested for tumors driven by mutations in the Shh pathway.…”

SGNE1/7B2 is epigenetically altered and transcriptionally downregulated in human medulloblastomas

“…The following primers were used: fw 5 0 -ccaagcttgacaatggtctccaggatg-3 0 , rev 5 0 -gggaattccctctggatccttatcctcatc-3 0 . The SGNE1-pcDNA4.1Myc/his expression vector was transfected into D283Med cells by nucleofection as described (Hartmann et al, 2006). After 12 h, transfected cells were selected with 400 mg/ml zeocin (Invitrogen).…”

“…Six hours before completion of the experiment, cells were pulse-treated with 3 H-thymidine (3 mCi/ml, Amersham, Munich, Germany). 3 H-thymidine uptake was determined as previously described (Hartmann et al, 2006) and normalized to the basal uptake in the control cells transfected with empty vector. (c) Representative image of a colony formation assay.…”

Embryonal neural tumours and cell death

“…During the last years it has become increasingly clear that activation of PI3K/Akt signalling is an essential anti-apoptotic event and PI3K/Akt directly targets a number of pro-and anti-apoptotic proteins (reviewed in [109]). A majority of both medulloblastoma and neuroblastoma primary tumours constitutively express activated Akt [22,23,110], and inhibition of PI3K/Akt signalling induces apoptosis of neuroblastoma cells [22,25]. Moreover, activated Akt significantly augments Hedgehog-induced medulloblastoma in mice and activation of PI3K/Akt signalling is important for the proliferation of cancer stem cells residing in the perivascular niche following radiation of medulloblastoma [111].…”