Inhibition of Phosphatidylinositol-3-OH Kinase/Akt Signaling Impairs DNA Repair in Glioblastoma Cells following Ionizing Radiation

Abstract

Radiation therapy is a mainstay in the treatment of glioblastomas, but these tumors are often associated with radioresistance. Activation of the phosphatidylinositol-3-OH kinase (PI3K)/Akt pathway, which occurs frequently in glioblastomas due to inactivation of the tumor suppressor phosphatase and tensin homologue (PTEN), correlates with radioresistance. To directly test the link between Akt activation and radioresistance, we utilized PTEN-deficient U251 glioblastoma cells engineered to inducibly restore PTEN upon exposure to doxycycline. These cells showed high basal levels of Akt activation (i.e. high levels of phospho-Akt), but induction of PTEN led to substantially decreased phospho-Akt and was associated with radiosensitization. To investigate whether the PTEN-induced radiosensitization was attributable to impaired sensing versus repair of DNA damage, we assessed levels of ␥-H2AX after ionizing radiation in U251 cells induced for PTEN. Initial post-radiation levels of ␥-H2AX foci were not decreased in PTEN-induced cells; however, the resolution of these foci was significantly delayed. In contrast to these results, induction of phosphatase-dead PTEN showed no appreciable effect. Finally, exposure of cells to the PI3K inhibitor LY294002 did not decrease the occurrence of ␥-H2AX foci after irradiation but did markedly delay their resolution. These results together support a direct link between Akt activation, repair of DNA damage, and radioresistance in glioblastoma. Targeting the PI3K/Akt pathway may modulate DNA repair to improve the efficacy of radiation therapy.Glioblastoma multiforme, the most common primary adult brain tumor, has a dismal prognosis. Even with aggressive surgery, radiotherapy, and chemotherapy, the median survival for patients with glioblastomas is under one year (1, 2). The phosphatidylinositol-3-OH kinase (PI3K) 3 signaling pathway is commonly activated in these tumors, often by virtue of PTEN gene mutation but possibly also by epidermal growth factor receptor expression (3, 4). PTEN encodes a phosphatase that dephosphorylates phosphatidylinositol-3,4,5 triphosphate to convert it to phosphatidylinositol-4,5 bisphosphate. Therefore, inactivation of PTEN leads to increased levels of phosphatidylinositol-3,4,5 triphosphate and increased Akt activation (5). Conversely, restoration of PTEN leads to inhibition of Akt. Chakravarti et al. (3) found significantly reduced survival times in patients whose tumors showed PI3K pathway activation. These patients were treated with a combination of surgery with postoperative radiation as the only adjuvant therapy, which suggested that this pathway might play an important role in radiation resistance.One of the factors implicated in radioresistance is activation of the Ras/PI3K/Akt pathway (6, 7). Data from numerous investigators show that inhibition of this pathway leads to radiosensitization, not just of glioblastomas but also carcinoma of the colon, bladder, prostate, head and neck, and cervix (6 -15).The precise mechanism by which the PI3K/Akt pathw...

PKB/Akt promotes DSB repair in cancer cells through upregulating Mre11 expression following ionizing radiation

“…We observed inhibition of Akt activity impaired the repair of radiation-induced DNA-DSB damage in CNE2 cells, whereas activated Akt promoted the repair of radiationinduced DNA-DSB damage in HeLa cells (Figure 1). These results were consistent with other recent studies which reported that activated Akt may decrease the efficacy of radiation therapy through promoting DNA repair in non-small-cell lung cancer cell lines and malignant-glioma cell lines (Tran et al, 2002;Kao et al, 2007;Toulany et al, 2008). Together, all these indicate that Akt can modulate radiation-induced DNA-DSB damage repair, and can regulate the efficacy of radiation therapy in other types of human cancers in which Akt kinase is hyperactivated.…”

“…g-H2AX, phosphorylation of the Ser residue (Ser139), usually appears rapidly and is recruited to megabase domains detectable as nuclear foci after exposure of cells to IR, which represents the sites of unrepaired DSBs. The number of g-H2AX correlates with the amount of unrepaired damage (Celeste et al, 2002;Aten et al, 2004;Kao et al, 2007), and a strong correlation between g-H2AX and cell death has been reported (Banath and Olive, 2003). Thus, g-H2AX foci assay has been regarded as a sensitive and specific assay for unrepaired DSBs (Taneja et al, 2004;Bao et al, 2006;Kao et al, 2007;Toulany et al, 2008).…”

“…Inhibition of phosphoinositide 3-kinase/Akt pathway leads to radiosensitization, such as glioblastomas, carcinoma of the breast, colon, bladder, prostate, head and neck and cervix (Gupta et al, 2002;Li et al, 2004;Altomare and Testa, 2005;Kim et al, 2005;Lee et al, 2006;Kao et al, 2007). One of the important reason is that activated Akt promotes survival of cells exposed to IR through inhibition of apoptosis (Brognard et al, 2001).…”

“…One of the important reason is that activated Akt promotes survival of cells exposed to IR through inhibition of apoptosis (Brognard et al, 2001). In recent studies, it was reported that phosphoinositide 3-kinase/ Akt pathway may modulate DNA repair to affect the efficacy of radiation therapy (Tran et al, 2002;Kao et al, 2007;Toulany et al, 2008). But the precise mechanism by which Akt activation regulates repair of DNA damage remains to be determined.…”

The pyridinylfuranopyrimidine inhibitor, PI-103, chemosensitizes glioblastoma cells for apoptosis by inhibiting DNA repair

“…Although the relationship between inhibition of PI3K signaling and increased DNA damage has been reported in the context of tumor radiosensitivity, increased either by LY294002 (Kao et al, 2007) or PI-103 (Prevo et al, 2008), the underlying molecular mechanism has so far not been investigated. Previously, distinct routes by which PI3K-mediated signaling could influence cellular DNA damage responses were suggested, such as interaction with DNA-PK-dependent DNA repair (Jackson, 2002).…”