The current standard of care for malignant glioma is initial treatment with radiation therapy combined with TMZ; however, malignant gliomas usually recur with a median time to progression of approximately 7 months . Two decades of molecular studies have identified important genetic events such as dysregulation of growth factor signaling via amplification or mutation of receptor tyrosine kinase genes; activation of PI3K pathway; and inactivation of p53 and Rb tumor suppressor pathways . In this study, we tried to identify the potential targets for counteracting the pro-survival signaling implicated in radioresistance of malignant glioma cells and to get insight into potential strategies to improve the therapeutic outcome of radiotherapy and TMZ in the management of GBM.
Inhibition of signal transduction pathways may provide the basis for a new paradigm of GBM therapy, based on the fact that most human gliomas exhibit aberrant activation of a pro-survival/pro-growth signaling network. EGFR is one of the most attractive therapeutic targets in GBM since the gene is amplified and over-expressed in approximately 40% of primary GBMs, especially those of the classical subtype. Nearly half of tumors with EGFR amplification also express a constitutively active EGFR mutant, EGF variant VIII (EGFRvIII), which has an in-frame deletion of exons 2–7 within the EGFR extracellular domain [16, 17]. Clinical trials with EGFR kinase inhibitors such as gefitinib and erlotinib did not show a significant benefit on overall survival or progression-free survival in patients with malignant glioma . Given the role of this growth factor receptor in gliomagenesis , the failure of EGFR inhibitors in GBM patients was particularly disappointing. Understanding the molecular mechanism of resistance may provide insight into the development of alternative strategies to tackle this issue.
Some studies found that tumors with EGFRvIII  and intact PTEN and tumors with low p-Akt levels are more likely to respond to EGFR inhibitors . Several investigators have identified loss of the PTEN tumor suppressor as a resistance factor for EGFR kinase inhibitor therapy [5, 20, 21]. Vivanco et al. also showed a critical role of PTEN in downregulation of activated EGFR. The PI3K/Akt/mTOR pathway is a critical regulator of tumor cell metabolism, growth, proliferation, and survival. In malignant gliomas, activity of this signaling network is frequently increased because of receptor tyrosine kinase over-activity, loss of PTEN tumor suppressor, and/or mutated oncogenic PI3K subunits . We observed a PTEN-mutant gloma cells showed higher radiosensitizing effect of PI103 than that of PTEN-wild type glioma cells. Our finding also supports the potential and rationale for PI3K targeting strategy in the treatment of malignant glioma having PTEN loss.
Attempts to inhibit the PI3K pathway with pan-PI3K inhibitors such as LY294002 have not progressed to clinical use due to concerns over organ toxicity and a lack of selectivity [22, 23]. Inhibition of the pathway using rapamycin resulted in paradoxical activation of Akt through loss of negative feedback in a subset of patients, which in turn was related to shorter time-to-progression during postsurgical maintenance rapamycin therapy . The limited single-agent activity of rapamycin analogs in several GBM trials [25, 26] provides a rationale for ongoing clinical trials with dual PI3K/mTOR inhibitors in GBM. A clinical trial of a dual PI3K/m-TOR inhibitor, XL765, in combination with TMZ is currently underway for GBM . Our results are in line with previous reports since combined treatment with TMZ and a dual PI3K/m-TOR inhibitor, XL765, has been successfully tested in glioma cell lines [23, 27]. Although rapamycin was a strong inducer of autophagy, it did not increased cytotoxicity of radiation therpy combined with temozolomide. In contrast, PI103 which is a dual inhibitor of class I PI3K and m-TOR prolonged gammH2AX foci formation with downregulation of p-DNA-PK, increased autophagy and increased cytotoxicity of radiation and temozolomide. We speculated that the impairment of DNA damage repair following radiation is potential mechanism of radiosensitization seen with this compound. Based on our results, we propose dual targeting PI3K/m-TOR with PI103 as a viable therapeutic strategy which should be explored to bypass the therapeutic resistance of GBM.
The data from TCGA for GBM indicate that tumorigenesis and progression involve multiple molecular abnormalities . HSP90, a molecular chaperon, is essential for the stability and function of many oncogenic client proteins that are frequently dysregulated in GBM, such as mutant EGFR, Akt, and p53. Since HSP90 is essential for the function of normal cells as well as tumor cells, one might be concerned that inhibition of its functions might not be selective for malignancy. Both preclinical and clinical observations, however, have shown that HSP90 inhibitors can be given in vivo at doses and schedules that exert antitumor activity without causing host toxicity . In addition to counteracting pro-survival signaling, HSP90 inhibitors block cell motility and invasion by suppressing multiple pro-invasive and pro-angiogenic cellular processes, such as MMP-2, VEGF  and EphA2 activity . Moreover, HSP90 can play a role in DSB repair and the activation of cell cycle check point . Inhibition of multiple signaling circuitries through the abrogation of HSP90 may be an effective treatment strategy for highly recalcitrant tumors such as GBM [33–35].
HDAC inhibitors (HDIs) target epigenetic modifications that interfere with transcriptional regulation and can induce growth arrest and cell death [36–38]. We previously reported that HDIs potentiate radiation-induced cell killing in a panel of human cancer cells through diverse mechanism: LBH589 preferentially radiosensitized human glioma cells that exhibited activated EGFR signaling due to the EGFRVIII mutation. Treatment with LBH589 led to acetylation of HSP90, which induced down-regulation of the client oncoproteins EGFR and decreased levels of p-Akt . LBH589 has also been reported to inhibit angiogenesis  and induce apoptosis, and delay of DNA damage repair in lung cancer cells with activated EGFR . Srivastava et al. reported that MS-275 inhibited tumor cell proliferation, angiogenesis, metastasis and reversing EMT in vivo breast cancer xenograft model by causing “cadherin switch” and decreased expression of VEGF, HIF-1, MMP-2 and MMP-9 . In our results, LBH589 significantly blocked migration, invasion, and vasculogenic mimicry formation through the down-regulation of VEGF, MMP-2, EphA2 and up-regulation of E-cadherin in U251 glioma cells. Given the link between mesenchymal feature and the progression of GBM, attacking EphA2, VEGF, and MMP-2 expression by these agents might be a potential strategy to improve the therapeutic outcome of combined radiotherapy and TMZ for GBM.