Treatment refractory glioblastoma (GBM) remains a major clinical problem globally, and targeted therapies in GBM have not been promising to date. The Cancer Genome Atlas integrative analysis of GBM reported the striking finding of genetic alterations in the p53 and PI3K pathways in more than 80% of GBMs. Given the role of these pathways in making cell-fate decisions and responding to genotoxic stress, we investigated the reliance of these two pathways in mediating radiation resistance. We selected a panel of GBM cell lines and glioma stem cells (GSC) with wild-type TP53 (p53-wt) and mutant TP53, mutations known to interfere with p53 functionality (p53-mt). Cell lines were treated with a brain permeable inhibitor of P-Akt (ser473), phosphatidylinositol ether lipid analogue (PIA), with and without radiation treatment. Sensitivity to treatment was measured using Annexin-V/PI flow cytometry and Western blot analysis for the markers of apoptotic signaling, alkaline COMET assay. All results were verified in p53 isogenic cell lines. p53-mt cell lines were selectively radiosensitized by PIA. This radiosensitization effect corresponded with an increase in DNA damage and a decrease in DNA-PKcs levels. TP53 silencing in p53-wt cells showed a similar response as the p53-mt cells. In addition, the radiosensitization effects of Akt inhibition were not observed in normal human astrocytes, suggesting that this treatment strategy could have limited off-target effects. We demonstrate that the inhibition of the PI3K/Akt pathway by PIA radiosensitizes p53-mt cells by antagonizing DNA repair. In principle, this strategy could provide a large therapeutic window for the treatment of TP53-mutant tumors. Mol Cancer Ther; 17(2); 336–46. ©2017 AACR.
See all articles in this MCT Focus section, “Developmental Therapeutics in Radiation Oncology.”
Glioblastomas (GBM) are among the most treatment-resistant solid tumors, often recurring after resection, radio-, and chemotherapy treatment. There has been a considerable effort to identify therapeutics that radiosensitize GBMs because most patients will receive radiotherapy. However, identifying such radiosensitive chemotherapeutic agents has been difficult due to the complex molecular heterogeneity of GBMs that promotes redundant pro-growth and pro-survival pathways. To overcome this obstacle, there is a need to devise therapeutic strategies targeting these redundant treatment-resistant pathways that promote the intrinsic radioresistance of GBMs.
The Cancer Genome Atlas (TCGA) reported that the PI3K and p53 pathways are each altered in over 80% of GBMs (1). Alterations in the PI3K pathway result in the constitutive activation of the signaling node Akt. High levels of Akt activation frequently results from the downregulation of the tumor-suppressor PTEN phosphatase or increased activity of upstream receptor tyrosine kinases (RTK; refs. 2–4). In GBMs, the most frequently upregulated RTKs are the EGFR, insulin-like growth factor 1 receptor beta (IGF1Rβ), and the platelet-derived growth factor receptor beta (PDGFRβ). The increased activity of Akt had been correlated with pro-growth and pro-survival factor. Conversely, p53 activity is suppressed in most GBMs. Decreased p53 activity predominantly stems from inactivating mutations or increased activity of the E3 ubiquitin-protein ligase (MDM2), a negative regulator of p53. Suppression of p53 activity alters cell fate decisions, DNA damage repair, apoptosis, and genetic stability. Given the high dysregulation of the PI3K and p53 pathways in GBMs and their role in responding to cellular stress, we set out to determine the interdependence of these two pathways in responding to radiation treatment.
In this study, we assess the radiosensitizing effect of inhibiting Akt activity in a panel of GBM cell lines with wild-type or mutant TP53. To inhibit Akt, we used the inhibitor phosphatidylinositol ether lipid analogue (PIA), which binds to the same region on the N-terminal Pleckstrin (PH) domain of Akt as PIP3 and blocks phosphorylation at serine 473 (5). Inhibitors competing for ATP-binding sites are common to many kinases, therefore, choosing to use PIA allows for greater specificity when targeting Akt (6, 7). We validate the results from our panel of GBM cell lines in p53 isogenic cell lines. Moreover, we investigate the role of DNA damage repair in the observed radiosensitizing effect of Akt inhibition in cell lines with wild-type or mutant TP53. Overall, we demonstrate that Akt inhibition by PIA before radiation treatment radiosensitizes GBM cells with mutant TP53.
Materials and Methods
This study was conducted in accordance with The Ohio State University Intuitional Review Boards for IRB (2009C0065 and 2014C0115), IBC (2009R0169), and IACUC (2009A0127).
Commercially available cell lines U87, LN229, and LN18 were purchased from the ATCC during 2015. Patient-derived cell lines OSU61, MGH8, VC3, and the GSC cell lines (OSU2GSC, OSU11GSC, and OSU20GSC) were authenticated by neuro-pathologists. The OSU cell lines were propagated during 2013 to 2015, MGH8 and VC3 during 2007 to 2008. Non-GSC cell lines were maintained in Dulbecco's Modified Eagle's medium (Invitrogen) supplemented with 10% FBS (Invitrogen) and 1% antibiotic–antimycotic (Invitrogen). GSCs were maintained in DMEM-F12 (Invitrogen) supplemented with B-27 (Gibco), EGF (20 ng/mL; Thermo Fisher), bFGF (20 ng/mL) (Thermo Fisher), and 1% antibiotic–antimycotic (Invitrogen). Normal human astrocytes (NHA) were obtained from Lonza and were propagated as per the manufacturer's protocol. All cells were cultured at 37°C under a gas phase of 95% air and 5% CO2 and were free from Mycoplasma contamination tested using a colorimetry-based assay (R&D systems) during the study period. All studies were conducted within 5 passages and were authenticated using STR profiles obtained from genetics core at University of Arizona.
Akt Inhibitor II (PIA) was purchased from Calbiochem. MK-2206 and PIK-75 were purchased from Selleckchem.
U87 cells were plated at a density of 2.5 × 105 cells per well in a 6-well plate. Media containing the lentivirus constructs for the Non-target or TP53 MISSION shRNA Lentiviral Transduction Particles (Sigma-Aldrich) were added at an MOI of 10. After 48 hours, the spent media were replaced with fresh media containing 10 μg/mL of puromycin (Sigma-Aldrich). Ten puromycin-resistant colonies were picked and each clone was expanded to assess the extent of p53 knockdown using RT-QPCR at transcriptional level and Western blot at translational level.
Cells were harvested using RIPA buffer (Sigma) with 1% (v/v) protease inhibitor cocktail (Sigma) and 1% (v/v) phosphatase inhibitor cocktail (Sigma). Samples were run on 10% SDS-PAGE gels and transferred to polyvinylidene difluoride membranes. The membranes were then incubated overnight with the following primary antibodies: EGFR, PDGFRβ, IGF1R, phospho-MDM2, phospho-p44/42 (P-ERK), p44/42 (ERK), PI3K-p85, MGMT, phospho-PTEN, PTEN, phospho-BAD, BAD, XIAP, phospho-mTOR, mTOR, ATM, phospho-Akt (Ser473), Akt, PARP, cleaved PARP, caspase-3, cleaved caspase-3, p38, phospho-p38, DNA-PKcs, Ku80, phospho-GSK3β, GSK3β, and γ-H2AX (Cell Signaling Technology). β-actin was purchased from Sigma-Aldrich.
Reverse-transcriptase quantitative polymerase chain reaction
Total RNA from clonal cells was isolated using the RNaseEasy spin columns (Qiagen) per the manufacturer's protocol. For reverse-transcriptase reactions, first-strand cDNA was synthesized using Superscript reverse transcriptase (Invitrogen) per the manufacturer's protocol. TaqMan probes (Applied Biosystems) were used to estimate the gene expression level of TP53 (Hs0015 3349_m1). GAPDH (Hs99999 905_m1) and RNA18S1 (Hs039 28990_g1) were used as housekeeping genes.
Clonogenic survival assay
Cells were plated at 8,000, 4,000, 2,000, 1,000, 500, and 50 cells per well in petri dishes and radiated at 2 Gy fractions totaling 10, 8, 6, 4, 2, and 0 Gy, respectively. The plates were incubated for 10 to 14 days depending on colony size and cell type. Colonies were stained with 0.1% methylene blue, dried, and counted. Colonies ≥50 cells were considered significant. The plating efficiency (PE) was calculated from the ratio of colonies formed over the number of cells plated (Fig. 2B). Radiobiological effect was quantified by computing radiation enhancement ratio (RER) at 2, 4, and 6Gy using survival fraction (SF) of RT alone over SF of PIA+RT (Fig. 2B) RER > 1 is indicative of radiosensitization.
To determine the number of apoptotic and necrotic cells, Annexin V/PI assays were performed using an apoptosis detection kit (Life Technologies). Briefly, cells were plated onto 6-well plates at a density of 2 × 105 cells per well and treated with PIA two hours before radiation treatment. After incubation, the cells were harvested and washed in cold PBS. For every 100 μL of sample, 5 μL of FITC Annexin V and 1 μL of 100 μg/mL PI were added and samples were incubated for 15 min at room temperature in the dark. The cells were then analyzed using flow cytometry and FlowJo software.
Alkaline comet assays
Alkaline comet assays were performed per manufacturer's instruction (Trevigen). Cells were trypsinized and suspended in cold PBS at a concentration of 1.0 × 105 cells/mL. The cells were then mixed with low melting agarose (Trevigen) at a ratio of 1:10 (v/v) and immediately plated onto Cometslide (Trevigen). Alkaline electrophoresis was run at 21 V for 30 minutes in the CometAssay Electrophoresis System (Trevigen). Data were collected using a fluorescence microscope (Zeiss).
In vivo studies
The glioblastoma xenograft model used has been outlined previously and the sample size for the study was determined on the basis of the power calculation from our previous experiments (8).
All results were confirmed in at least three independent experiments, and data from one representative experiment were shown. All quantitative data are presented as mean ± SD. The statistical analysis was performed using SAS 9.2 (SAS Institute) or GraphPad Prism 5. Student t tests were used for comparisons of means of quantitative data between groups. P values <0.05 were considered significant.
Basal expression of PI3K pathway proteins and MGMT in GBM cell lines
We selected a panel of commercially available GBM cells, patient-derived GBM cells, and glioma stem cell (GSC), containing five cell lines with mutant TP53 (p53-mt) and four cell lines with wild-type TP53 (p53-wt; Fig. 1A). MGH8 cells have a wild-type p53 sequence, whereas U87, VC3, OSU2GSC, and OSU11GSC cells have a mutation in the proline-rich domain leading to the amino acid change at residue 72 from proline to arginine. This P72R is a documented polymorphism and known to exhibit wild-type p53 function (9). LN229 have a mutation in the proline-rich domain P98L, this mutation has been reported to have a partial wild-type functionality (10). LN18, OSU61, and OSU20GSC have mutations in the DNA-binding domain of p53. Because PTEN is also an important regulator of Akt activity and apoptosis, we included the mutational status of PTEN to ensure observations based on the status of p53 were independent of PTEN (Fig. 1A). We began by determining the basal expression levels of the key proteins and regulators of the PI3K pathway (Fig. 1B–D). Akt was phosphorylated at serine 473 (P-Akt) in all cell lines, indicating activation of the PI3K pathway. U87, OSU61, and OSU20GSC showed relatively higher levels of P-Akt, a likely result of low PTEN expression. Conversely, the high levels of P-PTEN and low levels of PI3K in OSU2GSC and MGH8 cells likely account for the relatively low levels of P-Akt in these cell lines. All cell lines expressed the RTKs EGFR, PDGFRβ, and IGFR1β as well as the p53 inhibitor P-MDM2 (Ser166). In contrast to the GBM cell lines, normal human astrocytes (NHA) did not express any of the RTKs, Akt, or P-MDM2. The selected cell lines recapitulate the major genetic alterations of the PI3K and p53 pathways in GBM, and therefore were used to study the interdependent role of these two pathways in conferring radiation sensitivity. Next, we determined the basal levels of O6-methylguanine-DNA methyltransferase (MGMT), a predictive marker for response to temozolomide + radiotherapy (standard-of-care) in GBMs. The higher expression of MGMT in LN18, and OSU61 indicated that the MGMT promoter was likely unmethylated in these cell lines (Fig. 1C). Importantly, the expression of MGMT did not correlate with the status of p53, meaning observations relating to p53 will be independent of MGMT.
Mutational status of p53 determines the radiosensitizing effect of PIA treatment
The activation of Akt one hour after radiation treatment was observed in all cell lines (Fig. 1E), independent of p53 status, supporting the role of Akt in mediating radioresistance. Next, we characterized the sensitivity of U87 and LN18 cells to PIA treatment alone. PIA treatment-induced apoptosis in a time- and concentration-dependent manner (Fig. 1F; Supplementary Fig. S1). In addition, cells treated with greater than 5 μmol/L PIA showed a decreased colony formation ability (Supplementary Fig. S1). Interestingly, the colony formation was completely inhibited in LN18 after radiation treatment, whereas LN229 cells had colonies following radiation treatment, which may be explained on the basis of the partial wild-type functionality of p53 LN229. These results agree with a previous report that demonstrates PIA-induces a moderate level of apoptosis in cancer cell lines (11).
To determine whether PIA would radiosensitize GBM cells, we administered PIA 2 hours before radiation treatment. Cell lines with p53-mut (LN18, LN229, OSU61, and OSU20GSC) were radiosensitized by PIA, showing increased apoptosis and decreased colony forming ability (Figs. 2A and B, 3A; Supplementary Fig. S2). Clonogenic survival assay on the OSU61 cells and GSCs were not included because this cell line does not form colonies in a manner compatible with this assay. The cell lines with p53-wt (U87, MGH8, VC3, and OSU2GSC) were radioresistant to the addition of PIA, showing decreased apoptosis and increased colony forming ability (Figs. 2A and B, 3A; Supplementary Fig. S3). The plating efficiency (PE) and radiation enhancement ratio (RER) calculated are provided as tables accompanying Fig. 2B. As radiation dose increases RER increases in TP53-mt radiosensitive subset and decreases in the TP53-wt subset. Even at higher concentrations of PIA (50 μmol/L) U87 and LN18 cells followed the same radioresistant and radiosensitization trends, respectively (Supplementary Fig. S4). The radiosensitivity of these cell lines following Akt inhibition was independent of PTEN mutational status and MGMT expression. Representative microscopic images of LN18 (radiosensitive) and MGH8 (radioresistant) provide a snapshot of this differential radiosensitization (Fig. 2C). LN18 cells were more sensitive to PIA and radiation treatment, as indicated by the appearance of round, floating dead cells. Therefore, on the basis of these findings, we concluded that the radiosensitization of PIA might be dependent on the mutational status of p53.
Allosteric Akt inhibitor MK2206 radiosensitizes GBM cells independent of p53 status
To determine whether the radiosensitization effect of PIA was pathway or inhibitor specific, experiments were conducted using the Akt inhibitor MK-2206. MK-2206 binds and inhibits the phosphorylation of Akt at threonine 308 and serine 473 in a non-ATP competitive manner (12). Administration of MK-2206 and radiation did not show any selective radiosensitizing effects as seen with PIA (Supplementary Fig. S5). This differential effect between PIA and MK2206 could be due to the inhibition of Ser473 by PIA and both Ser473 and Thr308 by MK2206. Reports have shown the substrate specificity of DNA-PKc and Akt (Ser473) phosphorylation that further supports this conclusion (13). Further studies are required to confirm this DNA-PK–specific activity of Akt phosphorylation at Ser473 and Thr308 residues.
PIA and radiation treatment induce increased apoptosis in p53-mt cell lines
Western blots were run to determine the molecular targets by which PIA ± radiation treatments induce apoptosis. Following PIA and radiation treatments, the radiosensitive subset of cell lines had relatively higher levels of the apoptotic markers cleaved caspase-3 and cleaved PARP compared with the radioresistant cell lines (Fig. 3B). In addition, the radiosensitive cell lines showed relatively lower levels of the antiapoptotic proteins X-linked inhibitor of apoptosis (XIAP), survivin, and phosphorylated Bcl-2-associated death promoter (BAD; Fig. 3B). These alterations in apoptotic and antiapoptotic markers support the results of the Annexin-V/PI assays (Fig. 2A; Supplementary Fig. S2 and S3).
Furthermore, PIA ± radiation treatment reduced the activation of mTOR and Erk in all cell lines, corresponding with decreased cell proliferation (Supplementary Fig. S6). In contrast with a previous report, we did not observe the off-target activation of p38 after PIA treatment (7). However, we did observe an increase in p38 activity following PIA and radiation treatment in some cell lines (Supplementary Fig. S6). We do not expect that the activation of p38 is responsible for the observed radiosensitization effects as it is increased in both radiosensitive and radioresistant cell lines. In addition, the same report demonstrated that the induction of apoptosis after PIA treatment was independent of p38 activation. Since p73, like p53, is a tumor suppressor that induces cell-cycle arrest and participates in PTEN-induced apoptosis (14), we extended our investigation to determine the reliance of p73 on PIA-induced radiosensitization. Most the cell lines used in this study did not express p73. This is unsurprising given the p73 gene is in the commonly deleted chromosomal region 1p36.2-3. Furthermore, there was no change in PTEN expression following radiation or PIA ± radiation treatment (Fig. 3B). Therefore, we concluded that the alterations in apoptosis observed between the radiosensitive and radioresistance GBM cell lines were a result of Akt inhibition and the mutational status of p53 in these cell lines.
Validation of PIA-mediated radiosensitization by genetic approach
Five shRNA constructs were used to silence p53 expression in p53-intact U87 cells (Fig. 4A). U87 cells transduced with the p53 shRNA construct 1673 (U87-p53KD) did not have enhanced cell death following radiation treatment alone compared to the U87 non-target control cells (U87-NT; Fig. 4B). However, in the PIA ± radiation treatment arm, U87-p53KD cells experienced increased cell death (Fig. 4B). In addition, the U87-p53KD cells showed a decreased colony forming ability compared to U87-NT cells (Fig. 4C). The increased PIA and radiation-induced cell death in U87-p53KD cells were validated using the U87-p53KD 427 construct (Supplementary Fig. S7). Furthermore, U87-p53KD cells had an increased activation of the apoptotic markers cleaved caspase-3 and cleaved PARP, decreased Erk activation, and limited off-target p38 activation (Fig. 4D). These data support our previous findings that p53 may be a major determinant of radiosensitization following PIA treatment.
Normal human astrocytes are not radiosensitized by PIA
NHAs treated with PIA had 17% cell death and no increase in apoptotic or necrotic cell death following PIA and radiation treatment (Fig. 4E). This result could be due to the presence of functional p53 or the lower expression level of P-Akt in NHAs (Fig. 1B). Importantly, this suggests that normal tissue toxicity may be negligible if PIA-induced Akt inhibition is used in combination with radiation treatment in vivo. This is important because this could be a tumor-specific effect and provides a large therapeutic window and rationale for targeting P-Akt using PIA for the treatment of GBMs.
PIA and radiation treatment alters the DNA damage response in p53-mt cell lines
To determine the underlying cause of increased apoptosis in the cell lines radiosensitized by PIA treatment, we investigated the role of the cellular DNA damage repair response. The kinetics of radiation-induced DNA double-strand break repair (DNA-DSBR) through the non-homologous end joining (NHEJ) pathway have an ATM-independent fast and ATM-dependent slow component (15). Given that the expression of ATM following PIA ± radiation treatment did not show any trend specific to the radiosensitive or radioresistant cell lines (Supplementary Fig. S6), we directed our focus to the ATM-independent fast component of the NHEJ pathway. The fast component accounts for approximately 85% of DSBR and occurs within the first 2 to 3 hours following radiation treatment. The effect of PIA on double-strand DNA breaks (DSB) was estimated using a single cell gel electrophoresis COMET assay. This assay provides a qualitative way to compare DNA damage by observing tails of fragmented DNA behind cell nuclei. COMET assays performed on the radiosensitive LN18 and OSU61 cell lines following PIA treatment demonstrated a significant increase in DSBs in comparison to the U87 radioresistant cell line (Fig. 5A and B). Furthermore, p53 silencing in the U87-p53KD cell line had significantly higher amount of DSBs after PIA and radiation treatment (Fig. 5C). We concluded that this increase in DSBs following PIA ± radiation treatment may account for the enhanced cytotoxicity and radiosensitization of the GBM cell lines with functionally mutant or null p53.
Following radiation-induced DSBs, the fast component of the NHEJ pathways begins when the Ku70/80 heterodimer (Ku) binds the ends of DSBs and recruits DNA-dependent protein kinase catalytic subunits (DNA-PKc) that facilitate DNA repair. We found that PIA and radiation treatment of the radiosensitive GBM cell lines decreased DNA-PKcs expression in a time-dependent manner (Fig. 5D). Of note, comparing DNA-PKc expression levels after PIA + radiation treatment in LN18 and LN229 cells show a moderate decrease in DNA-PKc levels in LN229 cells compared to LN18 cells, likely due to the partial functionality of p53 (Fig. 5D). In contrast, the radioresistant GBM cell lines had increased DNA-PKcs expression following PIA and radiation treatment (Fig. 5E). U87-p53KD cells also had lowered expression of DNA-PKcs following PIA and radiation treatment compared with the U87-NT cells (Fig. 5E). Furthermore, OSU2GSC (TP53-wt; radioresistant) exhibited an increased DNA-PKc expression levels after PIA+RT and OSU20GSC (TP53-mt; radiosensitive) exhibited a decrease in DNA-PKc expression levels after PIA+RT (Fig. 5D and E). The modest change in GSK3β activation indicates limited off-target effects of PIA (Supplementary Fig. S8). In addition, minor changes in Ku80 suggest that the altered expression of DNA-PKcs are the driving factor behind differential DSBR. From these data, we concluded that the increased DNA damage in GBM cell lines with functionally mutant or null p53 likely results from the downregulation of DNA-PKcs.
In vivo studies conducted using U87-NT and U87-p53KD cells in NOD-SCID mice
Because of the higher intracranial tumor take rate, we used immunodeficient NOD-SCID mice. About half a million cells were intracranially implanted. The mice bearing U87-NT and U87-p53KD cells survived for 52 ± 5 days and 29 ± 5 days, respectively (Fig. 5F). On the basis of hematoxylin and eosin staining of coronal sections, it appears that silencing p53 leads to more aggressive tumors with an enhanced tumor growth kinetics and characteristics. We did not pursue in vivo studies further due to the growth kinetic alterations in the p53 isogenic cells, and owing to the loss of DNAPKc in NOD-SCID mice, and high sensitivity of these mice to radiation treatment.
PIK-75 ± PIA treatment radiosensitizes p53 intact GBM cell lines
Next, we investigated whether the dependence of p53-wt cells on NHEJ repair can be abrogated using the DNA-PKcs inhibitor PIK-75 (16). We determined the IC50 value of PIK-75 to be 0.5 μmol/L (Fig. 6A). Treating the p53-intact U87 and MGH8 cell lines with PIK-75 ± PIA radiosensitized these cell lines (Fig. 6B and C). In addition, we observed a higher susceptibility of U87 to the PIK-75 inhibitor in combination with radiation and PIA treatment. There appeared to be no colony formation at any radiation dose when U87 cells were treated with PIK-75 ± PIA (Fig. 6C). These data further support our hypothesis that PIA-induces radiosensitivity in cell lines with p53-mut by altering the expression of DNA-PKcs. In addition, this provides a therapeutic option for p53-wt GBM tumors.
Proof-of-concept from the previous study
A previous study (7) demonstrated that several commercially available lung and breast cancer cell lines had differential sensitivity to a panel of PIA inhibitors depending on the basal level of activated Akt in each cell line. They reported that PIAs increased apoptosis 20- to 30-fold in cancer cell lines with high levels of endogenous Akt activity but only 4- to 5-fold in cancer cell lines with low levels of Akt activity. All cell lines that showed higher sensitivity to PIA analogs were p53 mutated. We obtained sequencing data for H1703, H1155, and MB486 that had 1,009, 4,091, and 689 mutations, respectively. Among the cell lines used in our study, sequencing data for p53-mt cells LN18 and LN229 had about 949 and 563 mutations, respectively. We used these sequencing data to find out whether there was an association of any other mutations in all these cells lines in addition to p53 and apoptosis. By comparing the mutational profiles of p53-mt cell lines with high sensitivity to PIAs, we found that only the TP53 and PCLO genes were commonly mutated. In contrast, the radioresistant cell lines from our study shared no common mutations with the p53-wt cell lines which exhibited a decreased cytotoxicity to PIAs (Fig. 6D). Of note, U87 (radioresistant and p53-wt) also has the PCLO mutation, making TP53 the only commonly shared mutation unique to the radiosensitive group of cell lines and those cell lines with high susceptibility to PIAs. This further supports the correlation between the PI3K and p53 pathways in regulating survival in cancer cells.
Through this work, we aimed to clarify the interdependence of the PI3K and p53 pathways in conferring resistance to ionizing radiation in GBM cells. Inhibition of Akt by PIA would appear to be an effective radiosensitizer for GBMs, as summarized in the schematic in Fig. 6E. PIA is cell-permeable, reversible, and inhibits the activation of Akt with minimal off-target effects on PDK-1 or other kinases downstream of Ras, such as MAPKs (7). Because PIA is a lipid analog, it should have the ability to cross the blood-brain barrier, a major obstacle to the development of chemotherapeutics for gliomas. The efficacy and activity of PIA analogues have been validated in two different mouse models, proving that biologically effective doses of PIAs can be administered in vivo (17, 18). Furthermore, NHAs were not radiosensitized by PIA, suggesting that this treatment strategy may spare neighboring tissues from radiation induced-toxicity. Therapeutic targeting of several PI3K/Akt pathway members has been increasingly investigated and some of these inhibitors have made it to clinical trials (17, 19, 20). Some have tried to modulate the PI3K/Akt pathways by targeting upstream molecules such as Ras, which has been found to be activated in many tumor types (21–24). Others have tried to target the PI3K/Akt pathway by using PI3K inhibitors, such as wortmannin and LY294002, or mTOR inhibitors, such as rapamycin (5, 25). These inhibitors have been shown to reduce cancer cell growth in vitro and in vivo but have had limited success in clinical settings as single agents. Specifically, rapamycin's ability to induce Akt activity through upstream feedback loops has reduced its clinical antitumor ability (26). Furthermore, a number of feedback loops that activate Akt have been identified in tumor cells, making it more likely that targeting Akt directly may be a useful future cancer therapy approach (27). A preclinical pharmacology report correlating the activity of an Akt inhibitor to the genetic background of tumor cells supports the potential for using Akt inhibitors for personalized medicine based on genetic status (28). However, there have been conflicting reports from previous studies evaluating the use of P-Akt inhibitors as single agents or radiosensitizers for the treatment of glioma (29–32).
Previous reports demonstrate that Akt inhibitors induce cell death in cancer cell lines based on their basal level of P-Akt (4–7, 17, 19, 33, 34). In our study, PIA treatment induced apoptosis to a moderate level in p53-wt cell lines and to a greater extent in p53-mt cell lines. However, when combining PIA with radiation treatment, only cell lines with mutant p53 were radiosensitized by PIA. This observation was independent of PTEN mutational status as well as basal Akt activation. A case report on the molecular profiles of a glioma patient who survived for 20 years described that the tumor was PTEN positive (wild-type) and negative for P-Akt, giving support to the notion that this combination may have a favorable prognostic value (35). Therefore, the radiosensitization with PIA requires abrogation of “normal” p53 function/activity. Of course, we cannot completely exclude the possibility of other signaling molecules in modulating this effect. To account for this, at least in part, we have evaluated the expression levels of key off-target signaling proteins involved in the PI3K, apoptotic, and DNA-DSBR pathways. Furthermore, we found that the PIA-induced radiosensitization of the GBM cell lines tested were independent of other regulatory proteins in the p53 pathway, such as Murine double minute 2 (Mdm2) and CDKN2A. Mdm2 is a ubiquitin ligase that tags p53 for proteasome-mediated degradation. The level of P-Mdm2 expression has been reported to influence the extent to which radiation induces p53-dependent apoptosis (36). Basal P-Mdm2 levels were low in the GBM cell lines tested in this study (Fig. 1A) and did not correlate with either the radiosensitive or radioresistant group of cell lines following PIA treatment. CDKN2A, which encodes the MDM2 inhibitor p14ARF, was deleted in all cell lines used in this study. This supports our conclusion that p53 mutations that interfere with p53 wild-type functionality induce the radiosensitization effect of PIA.
The cell lines radiosensitized by PIA showed high levels of apoptosis that correlated with increased DNA damage and decreased levels of DNA-PKcs. There is a well-established link between Akt and DNA-PKcs, the major effector of processing and repair in the fast component of the NHEJ pathway. Akt binds to DNA-PKcs and is involved in facilitating binding to DNA damage sites and mediating the trans/auto-phosphorylation of DNA-PKcs, ensuring their release from DNA for further ligation of damaged ends (13, 37–41). The inhibition of Akt or DNA-PKcs is reported to induce high levels of DSBs and apoptosis after radiation treatment (21, 42–47). In contrast with Akt, the role of p53 in mediating radiation resistance in GBMs remains unclear. Interestingly, the inhibition of Akt by PIA was only sufficient to impair the DSBR and decrease DNA-PKcs expression in cell lines with functionally mutant or null p53. With regards to p53 status, we could only find one report on the pharmacological inhibition of p53-sensitizing GBM cells to BCNU and TMZ (48) and another report relating mutation of p53 and chemosensitivity in malignant gliomas (49). In spite of this, the link between DNA-PKcs and p53 is supported by an earlier finding that the defect in apoptosis in p53-deficient cells is rescued by the inactivation of the DNA-PK holoenzyme (50). This suggests that p53 may play a regulatory role in the DNA-PKcs–dependent response to DNA damage. Clinical studies attempting to seek a correlation between p53 status and radiosensitivity have provided mixed results (49) and constitute an area of improvement. On the basis of our results, we conclude that the inhibition of Akt in cell lines with functionally mutant or null p53 sensitizes GBM cells to radiation treatment by altering the DSBR. The main function of wild-type p53 is its tumor-suppressor activity, whereas mutant p53 acquire several functions to promote cancer phenotypes. Some of the p53 mutation confer loss of function and others that confer a change of function. The distinct functional classes of TP53 variants in cancers can lead to different consequences due to its regulation by an array of genetic and epigenetic alterations that occur in cancers which is beyond the scope of this study. However, in this study, the TP53 gene was sequenced in all the cell lines used and the interpretation of results were limited to the mutations described thereof and cannot be applied to all other TP53 disruptive or non-disruptive mutations that were not included. The findings of the study form a foundation for personalizing GBM therapies based on p53 status and supports the fact that, when using targeted therapies, treatment failure is primarily due to the compensatory effect of complicated gene and protein networks that allow cancer cells to evade death. As the paradigm of cancer treatment moves toward personalized care, using genetic aberrations in an individual patient's tumor to select treatments that maximize efficacy will be key for producing better treatment outcomes.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Conception and design: K. Palanichamy, S.-ei Noda, A. Chakravarti
Development of methodology: K. Palanichamy, A. Chakravarti
Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): K. Palanichamy, D. Patel, J.R. Jacob, K.T. Litzenberg, N. Gordon, S.-ei Noda
Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): K. Palanichamy, D. Patel, J.R. Jacob, N. Gordon, A. Chakravarti
Writing, review, and/or revision of the manuscript: K. Palanichamy, D. Patel, J.R. Jacob, K.T. Litzenberg, A. Chakravarti
Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): A. Chakravarti
Study supervision: K. Palanichamy, K. Acus, A. Chakravarti
NIH/NCI1RC2CA148190, RO1CA108633, and 1RO1CA188228 (to A. Chakravarti), and The Ohio State University Comprehensive Cancer Center and College of Medicine (to K. Palanichamy and A. Chakravarti).
We thank the OSU-CCC core facilities and all the patients enrolled in our IRB for making this possible. We thank Ananya Kamalakannan for her help with graphical abstract.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.