(2015) 34, 600–610 & 2015 Macmillan Publishers Limited All rights reserved 0950-9232/15 www.nature.com/onc

ORIGINAL ARTICLE TRIM24 promotes glioma progression and enhances chemoresistance through activation of the PI3K/Akt signaling pathway

L-H Zhang1, A-A Yin1, J-X Cheng, H-Y Huang, X-M Li, Y-Q Zhang, N Han and X Zhang

The tripartite motif TRIM24 (tripartite motif-containing 24) has been found to play distinct roles in tumor development and progression, according to different tumor contexts. However, it remains elusive whether TRIM24 plays a role in malignant gliomas that are the most common and deadly primary brain tumors in adults. We report here that TRIM24 expression is positively correlated with glioma malignancy and is negatively associated with prognosis of patients with newly diagnosed glioblastoma, which is the most malignant form of gliomas but displays highly heterogeneous clinical outcome. The multivariate Cox regression analysis demonstrates the independent predictive value of TRIM24 expression level for overall and progression-free survival. Knockdown of TRIM24 suppresses cell proliferation, cell cycle progression, clone formation and in vivo tumor development, whereas overexpression of TRIM24 promotes cell growth. Chromatin immunoprecipitation, real-time reverse –PCR and mutation analyses demonstrate that TRIM24 binds to the PIK3CA promoter via its PHD–Bromo domain to activate the transcription of PIK3CA , thus enhancing phosphatidylinositide 3-kinase (PI3K)/Akt signaling. The pan-PI3K inhibitor LY294002 and small interfering RNA targeting PIK3CA both abrogate the growth-promoting effect of TRIM24. Moreover, TRIM24 regulates the expression of DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT) through PI3K/Akt/nuclear factor-kB signaling transduction and enhances resistance to temozolomide, the standard chemotherapeutic agent for glioblastoma. Finally, glioblastoma patients with low TRIM24 expression benefit from chemotherapy, whereas those with high TRIM24 expression do not have such benefit. Our results suggest that TRIM24 might serve as a potential prognostic marker and therapeutic target for the management of malignant gliomas.

Oncogene (2015) 34, 600–610; doi:10.1038/onc.2013.593; publishd online 27 January 2014 Keywords: glioblastoma; TRIM24; TIF1a; PI3K; Akt; MGMT

INTRODUCTION In this study, we evaluated TRIM24 expression in gliomas, and In the brain and central nervous system, 80% of malignant tumors addressed a positive correlation between TRIM24 level and tumor are malignant gliomas, the majority of which are glioblastoma malignancy. TRIM24 promotes tumor growth and enhances multiforme (GBM, World Health Organization (WHO) grade IV resistance to chemotherapy via phosphatidylinositide 3-kinase astrocytoma).1 Despite great advances in surgery, chemotherapy (PI3K)/Akt signaling. Finally, possible clinical relevance of TRIM24 and radiotherapy, the median survival is only 12 to 15 months for effect on chemotherapy response was explored. patients with GBMs.2 This dismal outcome renders glioma an urgent subject of research. TRIM24 (formerly known as TIF1a) is the founding member of transcription intermediary factor (TIF) family3 that is characterized RESULTS by a conserved N-terminal tripartite motif (TRIM) consisting of a TRIM24 expression is positively correlated with tumor grade and RING domain, two B-box zinc-fingers and a coiled-coil region. In participates in GBM recurrence addition, its C-terminal tandem PHD finger and bromodomain Immunohistochemistry assay showed that normal brain glial and distinguish the TIF subgroup from other TRIM .4 oligodendroglioma presented negative TRIM24 staining or weak TRIM24 participates in chromatin remodeling,5 targets p53 for staining in few cells. However, TRIM24 was mostly expressed in degradation6 and interacts with nuclear receptors, either astrocytomas and was especially highly overexpressed in malig- positively or negatively, modulating the transcription activity of nant astrocytomas that include anaplastic astrocytoma (WHO these receptors.7 In recent years, close correlations of TRIM24 with grade III) and GBM (Figure 1a). The levels of TRIM24 expression several cancers have been unveiled. Nevertheless, its roles in presented a tumor pathological grade-dependent pattern, except cancers are seemingly controversial, such as acting as a tumor that no significant difference was observed between pilocytic suppressor in hepatocellular carcinoma8,9 while promoting astrocytoma (WHO grade I) and diffuse astrocytoma (WHO tumorigenesis in myeloid leukemia10,11 and breast cancer.12,13 grade II) (Figure 1b). This specific expression profile was further

Department of Neurosurgery, Xijing Hospital, Fourth Military Medical University, Xi’an, China. Correspondence: Professor X Zhang, Department of Neurosurgery, Xijing Hospital, Fourth Military Medical University, Changle West Road 127, Xi’an 710032, Shaanxi Province, China. E-mail: [email protected] 1These two authors contributed equally to this work. Received 30 April 2013; revised 14 October 2013; accepted 18 December 2013; publishd online 27 January 2014 TRIM24 in malignant gliomas L-H Zhang et al 601 (Figure 1d). Paired comparison found significant changes in TRIM24 levels before and after GBM recurrence (Po0.001).

TRIM24 level is inversely associated with prognosis of patients with newly diagnosed GBMs A total of 297 patients with newly diagnosed GBMs were included in the Cox proportional hazards regression models for determining prognostic factors. Mean value of Karnofsky performance score was 78.8, mean age at diagnosis was 60.3 and male-to-female ratio was 184:113. Because of diverse reasons, only 164 patients received chemotherapy whereas the others did not. The median overall survival (OS) among all newly diagnosed GBM patients was 11.3 months (95% confidence interval (CI), 10.8–11.8), whereas the median progression-free survival (PFS) was 5.7 months (95% CI, 5.3–6.1). The Kaplan–Meier estimates showed significant differences in both OS and PFS between patients whose tumors had low TRIM24 expression and those whose tumors had high expression (Po0.001 by the log-rank test; Figure 1e). The median OS among patients with low TRIM24 expression was 13.9 months (95% CI, 11.5–16.3) as compared with 11.0 months (95% CI, 10.6–11.4) among those with high expression. The median PFS for GBM patients with low and high TRIM24 expression was 6.5 months (95% CI, 5.5–7.5) and 5.3 months (95% CI, 4.8–5.8), respectively. Noteworthy, most of the long-term survivors (X36 months) presented low TRIM24 expression in their tumors (Figure 1f ). To further determine the prognostic value of TRIM24 ex- pression, we performed multivariate Cox proportional hazards regression analysis, incorporating standard clinical characteristics. The TRIM24 expression level, Karnofsky performance score and age emerged as significant independent prognostic factors for OS, and the former two factors also showed independent predictive value for PFS (Table 1).

TRIM24 promotes cell growth through PI3K/Akt signaling To determine potential mechanisms whereby TRIM24 associates with glioma grade and further negatively affects prognosis of GBM patients, we conducted a series of in vitro and in vivo assays by employing human GBM cell lines. Knockdown of TRIM24 by small hairpin RNA (shRNA) reduced the proliferation rates of T98 and U87 cells (Figure 2a). Cell cycle analysis confirmed that TRIM24 downregulation in T98 cells decreased the proportion of G2/M Figure 1. TRIM24 is overexpressed in astrocytomas, participates in phase, whereas forced overexpression of TRIM24 led to an GBM recurrence and is correlated with prognosis of GBM patients. increase in G2/M-phase proportion in U251 cells whose TRIM24 (a) Immunohistochemical staining of TRIM24 in surgical samples. AA, anaplastic astrocytoma; DA, diffuse astrocytoma; NB, normal levels are relatively lower (Figure 2b). Reduction of TRIM24 brain tissue; OG, oligodendroglioma; PA, pilocytic astrocytoma. Scale crippled soft agar colony formation ability of T98 and U87 cells, bar, 20 mm. (b) Comparison of TRIM24 levels among astrocytomas in manifested in both number and size of the clones (Figure 2c). different pathological grades. Error bars, s.d.; **Po0.01; NS, not In vivo experiments showed that once stably infected with shRNA significant. (c) Immunoblot analysis of TRIM24 protein levels in targeting TRIM24, T98 cells failed to form subcutaneous xenograft frozen preserved surgical samples. (d) Immunostaining of TRIM24 in even with the aid of Matrigel matrix, compared with the cells newly diagnosed GBM and recurrent GBM samples obtained from infected with nontargeting control shRNA (Figure 2d). To minimize the same patients. Shown are three representative recurrent GBM individual difference among mice, T98 cells infected with either cases that presented relatively low TRIM24 expression at the first shTRIM24 or shControl were inoculated into either flank of the surgery. Scale bar, 20 mm. (e, f) Kaplan–Meier estimates of prognosis same mice (Figure 2e). Again, TRIM24 knockdown abrogated in 297 newly diagnosed GBMs according to TRIM24 level. The differences in OS and PFS are both highly significant between tumor development, further validating the growth-promoting patients with low TRIM24 expression and those with high TRIM24 function of TRIM24 in vivo. expression (Po0.001 by the log-rank test). As activation of PI3K/Akt and Raf/MEK/extracellular-signal- regulated kinase (ERK) signaling are the main pathways participat- ing in GBM growth,14 we assessed levels of activated Akt and ERK, that represent their signaling status respectively. Knockdown of confirmed by western blot using proteins extracted from frozen TRIM24 by targeting shRNAs suppressed phosphorylation of Akt preserved tissue samples (Figure 1c). (Figures 3a and b). Concomitantly, the phosphorylation of ERK For recurrent GBM cases, compared with their first surgical increased, probably reflecting that the inhibition effect of samples according to which GBM was newly diagnosed, TRIM24 activated Akt on Raf/MEK/ERK signal transduction was relieved.15 expression generally increased at the time of disease recurrence, The impact of TRIM24 knockdown on cell signaling was rescued especially in those who showed low expression previously by reintroduction of TRIM24 into downregulated cells by

& 2015 Macmillan Publishers Limited Oncogene (2015) 600 – 610 TRIM24 in malignant gliomas L-H Zhang et al 602 Table 1. Cox proportional hazards regression analysis on newly diagnosed GBMs

Variable Progression-free survival Overall survival

Univariate Cox analysis Multivariate Cox analysis Univariate Cox analysis Multivariate Cox analysis

P-value HR (95% CI) P-value HR (95% CI) P-value HR (95% CI) P-value HR (95% CI)

TRIM24 expression o0.001 1.50 (1.32–1.71) o0.001 1.43 (1.26–1.63) o0.001 1.60 (1.39–1.83) o0.001 1.50 (1.30–1.72) KPS o0.001 0.98 (0.97–0.99) 0.001 0.98 (0.97–0.99) o0.001 0.98 (0.97–0.99) 0.002 0.98 (0.97–0.99) Age 0.029 1.01 (1.00–1.03) 0.093 1.01 (1.00–1.02) 0.001 1.02 (1.01–1.03) 0.012 1.02 (1.00–1.03) Sex 0.572 1.07 (0.84–1.36) 0.201 1.17 (0.92–1.49) 0.887 0.98 (0.77–1.25) 0.891 1.02 (0.80–1.30) Extent of resection 0.144 0.87 (0.72–1.05) 0.074 0.85 (0.70–1.02) 0.418 0.93 (0.77–1.11) 0.204 0.89 (0.75–1.07) Chemotherapy 0.007 0.72 (0.57–0.92) 0.095 0.82 (0.64–1.04) 0.001 0.67 (0.53–0.85) 0.088 0.81 (0.63–1.03) Abbreviations: CI, confidence interval; GBM, glioblastoma multiforme; HR, hazard ratio; KPS, Karnofsky performance score.

Figure 2. TRIM24 promotes tumor growth. (a) Proliferation assay of T98 and U87 cells infected with retrovirus encoding shRNA targeting TRIM24 or nontargeting control shRNA. Error bars, s.e. (b) Cell cycle analysis of T98 and U251 cells. T98 cells were infected with retrovirus encoding shRNA against TRIM24 and negative control shRNA, whereas U251 cells were infected with lentivirus containing human TRIM24 ORF or empty control lentivirus. Error bars, s.e. (c) Soft agar clone formation assay of T98 and U87 cells infected with shTRIM24 or shControl retrovirus. Representative images are shown. Scale bar, 200 mm. (d, e) Xenograft tumor formation of T98 cells infected with shTRIM24 or shControl retrovirus.

Figure 3. TRIM24 activates PI3K/Akt signaling through enhancing PIK3CA expression. (a) Immunoblot analysis of parental T98 cells and the cells infected with retrovirus encoding shRNAs targeting TRIM24 or nontargeting control shRNA. (b) Immunoblot analysis of parental U87 cells and the cells infected with shTRIM24 or shControl. Rescue of TRIM24 expression in cells previously infected with shTRIM24 retrovirus was performed by using lentivirus containing human TRIM24 ORF or empty control lentivirus at a multiplicity of infection value of 4, and 4 days after reintroduction of TRIM24, cell lysates were collected. (c) T98 cells were infected with retrovirus encoding shTRIM24 or shControl, or infected with lentivirus expressing TRIM24 or empty control lentivirus. After 3 days, 2 mM LY294002 or an equal volume of DMSO was added and MTT assay was performed for 4 consecutive days. The inhibiting efficiency of LY294002 on PI3K/Akt signaling was tested by western blot. Error bars, s.e. (d) Real-time RT–PCR analysis of PIK3CA in T98 and U87 cells infected with shTRIM24 or shControl retrovirus. Error bars, s.e. (e) ChIP analysis of the PIK3CA promoter. After coimmunoprecipitation with TRIM24 antibody or control immunoglobulin G (IgG), the DNA was PCR amplified using primers corresponding to five different sites and resolved in 2% agarose gels. (f) T98 cells were infected with shTRIM24 or shControl. Rescue of TRIM24 expression in cells previously infected with shTRIM24 retrovirus was performed by transfection using pcDNA3.1( þ ) containing full-length TRIM24 ORF or mutant TRIM24 deleted of the PHD–Bromo domain, and 500 mg/ml Geneticin selection was employed to increase the efficiency of rescue. After 2 weeks, cell lysates were collected for immunoblot analysis. Wild-type TRIM24 rescues PIK3CA and p-Akt levels, whereas mutant TRIM24 has no significant effect. D, deletion of PHD–Bromo; Error bars, s.e.; **Po0.01; NS, not significant. (g) ChIP analysis of the PIK3CA promoter in differently treated T98 cells.

Oncogene (2015) 600 – 610 & 2015 Macmillan Publishers Limited TRIM24 in malignant gliomas L-H Zhang et al 603 infection with lentivirus containing human TRIM24 open reading Then, we hypothesized that the growth of TRIM24-down- frame (ORF), whereas empty control lentivirus had no effect regulated cells should be less sensitive to the inhibitor of PI3K/Akt (Figure 3b). transduction because the signaling has already been suppressed

& 2015 Macmillan Publishers Limited Oncogene (2015) 600 – 610 TRIM24 in malignant gliomas L-H Zhang et al 604 therein, whereas its influence on TRIM24-overexpressed cells weights of developed tumors. In addition, downregulation of should be more significant because of the augmented signaling TRIM24 enhanced the therapeutic effect of TMZ administration activation. This hypothesis was tested by employing LY294002, (Figure 4f) and the additive effect was significant (Po0.01), as a specific inhibitor of PI3K/Akt signaling.16 Relative effect of determined by factorial analysis. LY294002 was evaluated, by normalizing to dimethyl sulfoxide TMZ-induced injury can be repaired by O6-methylguanine-DNA (DMSO) that was used to dissolve LY294002. Inhibition of Akt methyltransferase (MGMT), a DNA repair enzyme whose key role activation had less effect on TRIM24-downregulated cells as in TMZ resistance is now commonly recognized.19 Thus, we compared with the control cells (Figure 3c). The effect of inhibition determined possible change in MGMT expression level after was amplified in TRIM24-overexpressed cells as compared with TRIM24 knockdown. After infection with shRNAs targeting TRIM24, the cells infected with control lentivirus. In other words, inhibition the cells expressed less MGMT as compared with parental cells of Akt phosphorylation abrogates the growth-promoting effect of and cells infected with shControl (Figure 5a). Real-time RT–PCR TRIM24 (Figure 3c and Supplementary Figure S1). By the way, analysis demonstrated that TRIM24 knockdown reduced MGMT in LY294002 induces similar effects on Akt and ERK phosphorylation the transcriptional level (Figure 5b). However, we did not observe as TRIM24 knockdown (Figure 3c). To evaluate whether TRIM24 TRIM24 binding to the MGMT promoter by employing ChIP assay regulates Akt activation in glioma tumors, immunoblot assay was (data not shown), using primers described previously.20 employed to test the proteins in surgical samples and a significant Nevertheless, TRIM24 regulates PI3K/Akt signaling that activates correlation (Po0.001) was observed between TRIM24 and p-Akt nuclear factor (NF)-kB, and NF-kB has binding sites within the levels in GBMs (Supplementary Figure S2). MGMT promoter. To test this hypothesis, we employed a luciferase The levels of PI3K/Akt signaling components were then reporter construct containing NF-kB response elements and found evaluated as described.17,18 Real-time reverse transcription–PCR that NF-kB activity was reduced upon TRIM24 knockdown (RT–PCR) and immunoblot analyses demonstrated the reduction (Figure 5c). Similar results were observed when cells were treated of PIK3CA levels upon TRIM24 knockdown (Figures 3d and f), with LY294002 (data not shown). Moreover, the effect of TRIM24 whereas no significant change was observed in other compo- knockdown on MGMT expression was rescued by reintroduction nents. Besides, knockdown of PIK3CA using targeting small of TRIM24 into downregulated cells by infection with lentivirus interfering RNA (siRNA) had similar effects as the pan-PI3K containing TRIM24 ORF, whereas empty control lentivirus had no inhibitor LY294002 on the growth-promoting effect of TRIM24 effect. Transfection of siRNA targeting RelA, which encodes a (Supplementary Figure S3). Furthermore, chromatin immuno- subunit of NF-kB, abolished the rescue effect of TRIM24 precipitation (ChIP) assay demonstrated TRIM24 binding to the (Figure 5d). PIK3CA promoter in T98 and U87 cells (Figure 3e). As TRIM24 Because MGMT consumption takes place during the DNA repair recognizes histone markers by means of its PHD–Bromo domain,12 process,21 we hypothesized that apart from affecting baseline level rescue experiments were performed in TRIM24-downregulated of MGMT, TRIM24-depleted cells might fail to replenish enough cells. Wild-type TRIM24 rescued PIK3CA and p-Akt levels, MGMT to counteract insistent TMZ exposure, and this consequence whereas mutant TRIM24 deleted of the PHD–Bromo domain could appear more pronounced as the intensity of TMZ action had no effect (Figure 3f). The binding on the PIK3CA promoter was escalated. Our hypothesis was tested by exposing cells to TMZ at abrogated when the mutant TRIM24 was introduced into TRIM24- increased concentrations for 1 week, a period enough for the downregulated cells (Figure 3g). However, there was still some consumption of preliminarily preserved MGMT. As expected, the sort of residual binding to the PIK3CA promoter, possibly because decrease in MGMT levels presented a TMZ dose-dependent manner the elimination of wild-type TRIM24 could not be complete by in TRIM24-depleted cells, with the lowest MGMT level in the highest RNA interference. TMZ concentration group. Nevertheless, no significant change in MGMT level was observed in control cells wherein MGMT replenish capacity retained (Figure 5e). This specific tendency also manifested TRIM24 enhances resistance to temozolomide (TMZ) itself in the plate clone formation assay (Figure 4b). Besides, Chemotherapy resistance is a major cause for failed GBM significant correlation (Po0.01) between TRIM24 and MGMT levels treatment with alkylating agents, among which TMZ is considered was observed in GBMs (Supplementary Figure S4). to be standard.2 To investigate whether TRIM24 would affect cell response to TMZ, we cultured the cells, infected with either shTRIM24 or shControl, in the presence of TMZ. Considering Low TRIM24 expression is a prerequisite for effective that TRIM24 knockdown alone had impact on cell growth, we chemotherapy employed the relative viability (TMZ treatment group normalized In light of TRIM24 effect on chemosensitivity in lab assays and to DMSO group for either infected cells), so as to eliminate false nearly half of GBM patients in this study did not receive positive confounding. MTT assay showed that downregulation of chemotherapy, we thus attempted to explore the impact of TRIM24 increased the cytotoxicity of TMZ in T98 and U87 cells TRIM24 level on chemotherapy in the clinical context. To this end, (Figure 4a). Plate clone formation assay demonstrated that TMZ GBM patients were subdivided according to TRIM24 expression treatment had additive effect on TRIM24 knockdown that alone level in their tumors and the therapeutic effect of chemotherapy led to decreased colonies (Figure 4b). Exposure to TMZ for 1 week was separately analyzed. Interestingly, a significant impact of resulted in significant cleavage of caspase-7 in TRIM24-depleted TRIM24 on chemotherapy response was noticed. That is, in the low T98 cells, whereas minimal effect was observed in the control cells TRIM24 expression subgroup, patients who received chemo- (Figure 4c). TUNEL (TdT-mediated dUTP nick end labeling) and therapy had a median OS of 14.8 months as compared with 9.5 flow cytometry analyses showed that depletion of TRIM24 alone months for those who did not receive chemotherapy (P ¼ 0.008; did not initiate apparent cell . Nevertheless, apoptosis Figure 6a). Moreover, in this subgroup, those who received emerged evidently when TRIM24 depletion was combined with chemotherapy had a median PFS of 6.6 months as compared TMZ treatment (Figures 4d and e). with 5.1 months for those who did not receive chemotherapy To investigate whether TRIM24 downregulation would affect (P ¼ 0.042; Figure 6b). On the other hand, among the patients tumor chemosensitivity in vivo, U87 cells were subcutaneously whose tumors displayed high TRIM24 expression, no significant inoculated into nude mice after infection with shRNAs and TMZ prognostic benefit from chemotherapy was observed (Figures 6c dissolved in saline was orally administered. Similar to the results and d). Besides, the multivariate Cox regression analysis confirmed obtained from T98 cells, which failed to form xenograft if TRIM24 the independent role of TRIM24 expression in chemotherapy was depleted, knockdown of TRIM24 in U87 cells reduced the response.

Oncogene (2015) 600 – 610 & 2015 Macmillan Publishers Limited TRIM24 in malignant gliomas L-H Zhang et al 605

Figure 4. TRIM24 enhances resistance to TMZ. (a) T98 and U87 cells were infected with retrovirus encoding shTRIM24 or shControl. After 3 days, TMZ or an equal volume of DMSO was added and MTT assay was performed for four consecutive days. Error bars, s.e. (b) Plate clone formation assay of T98 cells infected with shTRIM24 or shControl. Cultures were exposed to TMZ in different concentrations or DMSO in equal volumes. After 2 weeks, the colonies containing more than 50 cells were recorded. Error bars, s.e.; *Po0.05. (c) Immunoblot analysis of T98 cells treated with 500 mM TMZ for 1 week. (d) TUNEL assay of T98 cells treated with 500 mM TMZ for 1 week. Hoechst 33258 was used to counterstain all nuclei. Scale bar, 100 mm. (e) Flow cytometry using Annexin V–fluorescein isothiocyanate (FITC) and propidium iodide (PI) double staining was employed to sort treated T98 cells in apoptotic phase. Positive Annexin V staining represents cell apoptosis. Error bars, s.e.; **Po0.01; NS, not significant. (f) Nude mice were inoculated with U87 cells infected with shTRIM24 or shControl. TMZ was orally administrated while saline was used as control. Developed tumors were weighted. Error bars, s.d.; **Po0.01.

DISCUSSION approaches, whereas the cells with low expression are gradually Patients suffering from malignant gliomas display heterogeneous replaced or eradicated by treatments. clinical outcome, especially among GBM patients, although their The effects of TRIM24 on GBMs manifest in tumor growth tumors appear histologically similar.22 Identification of tumor and chemoresistance. The growth-promoting effect of TRIM24 biomarkers can aid in prognostic prediction and modulation of emanates through PI3K/Akt signaling pathway that is often conventional therapies, as well as molecular targeting approaches. overactivated in GBMs to stimulate cell proliferation and inhibit In this study, we discovered that the expression of TRIM24 not apoptosis.23 TRIM24 also promotes breast cancer development as only presented a tumor grade-dependent pattern but also a potent of -a,12 and it also enhances distinguished patients with good prognosis from those with prostate cancer progression by activating androgen receptor.24 On poor outcome within a single grade-GBM, suggesting that TRIM24 the other hand, TRIM24 suppresses hepatocarcinogenesis by detection may improve the predictive strength of currently inactivation of -a8,25 that affects interferon/ available prognostic indicators. However, we could not address signal transducer and activator of transcription (IFN/STAT) the relationship of TRIM24 expression and prognosis of patients pathway.26,27 TRIM24 also prevents arterial calcification by with gliomas in other pathological grades because of not enough suppressing the vitamin D receptor signaling pathway.28 These prognostic information about them. The upregulation of TRIM24 conflicting findings might be because of the switch of TRIM24 at GBM recurrence might imply the contribution of evolutional roles between activator and suppressor according to different pressure, because overexpressed cells may possess greater cellular pathways, and what signaling is pivotal in a specific proliferation rate and circumvent conventional therapeutic disease, thus ultimately dictating in which direction the balance

& 2015 Macmillan Publishers Limited Oncogene (2015) 600 – 610 TRIM24 in malignant gliomas L-H Zhang et al 606

Figure 5. TRIM24 regulates MGMT expression via activation of NF-kB. (a) Immunoblot analysis of parental T98 cells and the cells infected with shTRIM24 or shControl retrovirus. (b) Real-time RT–PCR analysis of MGMT gene expression in T98 cells infected with shTRIM24 or shControl retrovirus. Error bars, s.e.; **Po0.01. (c) Cells were co-transfected with retroviral plasmids encoding shRNAs, luciferase reporter construct containing NF-kB response elements and pRL-TK vector. The NF-kB activity decreases upon TRIM24 knockdown. Error bars, s.e.; *Po0.05; **Po0.01. (d) Immunoblot analysis of T98 cells infected with shTRIM24 or shControl. Rescue of TRIM24 expression in cells previously infected with shTRIM24 retrovirus was performed by using lentivirus containing human TRIM24 ORF or empty control lentivirus at a multiplicity of infection value of 2 for 8 h. Targeting RelA siRNAs or nontargeting control siRNAs were transfected as needed after cells were recovered from lentivirus infection overnight. Additional 2 days later, cell lysates were collected for immunoblot analysis. Real-time RT–PCR was employed to validate the rescue of TRIM24 gene expression and knockdown efficiency of siRNA. Knockdown of RelA abrogates the rescue effect of TRIM24 on MGMT expression. (e) After infection with shTRIM24 or shControl retrovirus, T98 cells were exposed to TMZ in different concentrations for 1 week. TMZ consumes MGMT and TRIM24-depleted T98 cells fail to replenish it efficiently, whereas MGMT levels retain in control cells. Error bars, s.e.; **Po0.01.

will be tipped. For example, administration of retinoid acid T98 and U87 cells might be attributed to their p53 status, TRIM24 induces proliferation of normal hepatocyte,29 whereas adaptive knockdown efficiency and, of course, MGMT expression profile.35 mechanism that is activated to fight against hyperproliferation can TRIM24 binds to the PIK3CA promoter via its PHD–Bromo lead to increased ploidy that is prone to initiate carcinogenesis in domain and induces the overexpression of PIK3CA that in the liver.26 However, activation of retinoic acid receptor-a in GBMs turn augments the phosphorylation of Akt. Phosphorylated Akt indicates longer survival for patients.30 In addition, TRIM24 has activates MDM236 that promotes p53 degradation.37 Therefore, been found to be a negative regulator of the tumor suppressor besides direct interaction with p53, TRIM24 might indirectly p53 in embryonic stem cells and several cancer cell lines,6,31 but regulate p53 level through PI3K/Akt signaling. As Ras activates the p53 regulation mechanism may be different in liver. For PI3K38 whereas p-Akt inhibits Raf,36 the increased phosphorylation instance, irradiation cannot induce p53 accumulation in the liver,7 of ERK could reflect that the inhibition effect of activated Akt on and MDM2 loss does not alleviate the instability of mutant p53 in Raf/MEK/ERK signal transduction was relieved. In addition, the liver, whereas it does so in the brain.32 Nevertheless, TRIM24 considering that TRIM24 knockdown inhibits cell proliferation, seems to have p53-independent effect in gliomas, because U87 the changes in ERK activation are more likely to be secondary and cells express wild-type p53 whereas T98 and U251 cells express compensatory, indirectly demonstrating that TRIM24 acts on PI3K/ mutant p53, similar to the results obtained from lung cancer.33 Akt signaling via regulating PIK3CA expression (Figure 7). Knockdown of TRIM24 in T98 and U87 cells increased the levels of Apart from its role in tumor growth, TRIM24 enhances p53 protein (Supplementary Figure S5). However, the baseline resistance to alkylating chemotherapy by regulating the DNA level and induced level of p53 were both higher in T98 cells than repair enzyme MGMT that removes alkyl groups from the O6 those in U87 cells, owing to its mutant form in the former.34 position of guanine and blunts the cytotoxicity induced by Therefore, the different sensitivity to TMZ-induced cytotoxicity in chemotherapy.39 Our results suggest that TRIM24 regulates MGMT

Oncogene (2015) 600 – 610 & 2015 Macmillan Publishers Limited TRIM24 in malignant gliomas L-H Zhang et al 607

Figure 6. Kaplan–Meier estimates of OS and PFS of newly diagnosed GBM patients according to TRIM24 level and chemotherapy status. (a)In the low-TRIM24 expression group (n ¼ 83), those who received chemotherapy had a 48% risk reduction in death (hazard ratio (HR), 0.52; 95% CI, 0.32–0.85), as compared with those who did not receive chemotherapy. (b) In the low-TRIM24 expression group, chemotherapy yielded a 38% risk reduction in tumor progression (HR, 0.62; 95% CI, 0.39–0.99). (c, d) Minimal but not significant therapeutic effect of chemotherapy was observed among patients whose tumors displayed high TRIM24 expression.

chemotherapeutic agents. Moreover, GBM patients with low TRIM24 expression have significant response to chemotherapy, whereas those with high TRIM24 expression do not, suggesting that evaluation of TRIM24 levels in surgical samples might offer a route to stratify patients for achieving efficient individual therapy. Nevertheless, longitudinal studies with a larger and broader population are needed for validation. In conclusion, these findings indicate that TRIM24 promotes glioma progression and enhances chemoresistance in GBMs via PI3K/Akt signaling. Therefore, TRIM24 might have prognostic and therapeutic implications for the management of malignant gliomas.

MATERIALS AND METHODS Patients and tissue samples Surgical samples were collected at the Department of Neurosurgery, Xijing Figure 7. Schematic diagram depicting TRIM24-regulated pathways Hospital between 2005 and 2010. They included normal brain tissue in glioma cells. TRIM24 activates PI3K/Akt signaling that subse- (n ¼ 15), pilocytic astrocytoma (WHO grade I; n ¼ 24), diffuse astrocytoma quently elevates NF-kB activity. Activated NF-kB translocates into (WHO grade II; n ¼ 57), anaplastic astrocytoma (WHO grade III; n ¼ 63), the nucleus and binds to the MGMT promoter, thus enhancing its newly diagnosed GBM (WHO grade IV; n ¼ 297), recurrent GBM (n ¼ 48) transcription. Upregulation of the PI3K/Akt pathway promotes cell and oligodendroglioma (n ¼ 44).40 All patients provided written informed proliferation and inhibits apoptosis induced by chemotherapy, consent and this study was approved by the Institutional Review Board. whereas MGMT only affects the latter. The crosstalk exists between PI3K/Akt and Ras/Raf/MEK/ERK pathways. Besides direct interaction with p53, TRIM24 may have indirect effect through regulating Immunohistochemistry MDM2. Formalin-fixed, paraffin-embedded tissues were employed for immuno- histochemistry with anti-TRIM24 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and nuclei were slightly counterstained with expression through PI3K/Akt/NF-kB signaling transduction. As hematoxylin. The intensity and percentage of positive cells were evaluated TRIM24 is overexpressed in GBMs, especially those leading to in at least five separate fields at  400 magnification. The scores were poorer prognosis, TRIM24 may be considered as an attractive evaluated by two pathologists who were blinded to clinical data. therapeutic target alone or in combination with conventional Immunoreactivity was scored as follows: 0, no staining; 1, weak staining

& 2015 Macmillan Publishers Limited Oncogene (2015) 600 – 610 TRIM24 in malignant gliomas L-H Zhang et al 608 in o50% cells; 2, weak staining in X50% cells; 3, strong staining in o50% per week. After 2 weeks, colonies larger than 50 mm in diameter were cells; and 4, strong staining in X50% cells. recorded in five separate fields.

Western blot Plate clone formation assay Tissue and cell samples were lysed in RIPA buffer supplemented with 1 mM For clone formation, 150 cells were seeded in 60 mm dishes after infection NaF, 1 mM Na3VO4 and protease inhibitor cocktail (Roche Diagnostics, with shRNAs. After cell attachment, cultures were exposed to TMZ in Mannheim, Germany). Equal amounts of total protein were resolved by different concentrations. After 2 weeks, cells were stained with 0.25% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and trans- crystal violet and the colonies containing more than 50 cells were ferred to nitrocellulose membranes. The primary antibodies against recorded. TRIM24, PIK3CA, Actin (Santa Cruz Biotechnology), p-Akt (Ser 473), Akt, p-ERK1/2, ERK1/2, procaspase-7, cleaved caspase-7 (Cell Signaling Techno- logy, Beverly, MA, USA), MGMT, p53 (Bioworld Technology, St Louis, MO, TUNEL assay USA) were used according to the manufacturers’ recommendations. Each After treatment, cells grown in the glass bottom dishes were fixed with 4% immunoblot was done at least thrice and the signals were quantified using paraformaldehyde and permeabilized with 0.1% Triton X-100. The DNA ImageJ software (Bethesda, MD, USA). strand breaks in apoptotic cells were labeled with the TUNEL reaction mixture (Roche Diagnostics) and then all nuclei were counterstained with Hoechst 33258. Dishes were photographed under a Fluoview FV10i Cell culture and drugs confocal fluorescent microscope (Olympus, Shinjuku, Tokyo, Japan). The human GBM cell lines T98, U87 and virus packing cell lines PT67, 293T were obtained from the ATCC (Manassas, VA, USA). Cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal Real-time RT–PCR Total RNA was isolated with RNAiso Plus (Takara, Dalian, China) and reverse bovine serum at 37 1Cin5%CO2. TMZ (Sigma-Aldrich, St Louis, MO, USA) was dissolved in DMSO (Sigma-Aldrich) at a stock concentration of transcribed with PrimeScript RT Master Mix (Takara). PCR amplification was 100 mmol/l and stored at À 20 1C. For in vivo studies, TMZ (Schering- performed in triplicate with SYBR Premix Ex Taq II (Takara) using CFX96 Plough, Kenilworth, NJ, USA) was dissolved in saline (4 mg/ml) for oral Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). The values administration. A 10 mM solution of LY294002 in DMSO was purchased were normalized to the levels of glyceraldehyde-3-phosphate dehydro- from Merck Biosciences (Darmstadt, Germany). Puromycin and polybrene genase (GAPDH). Part of the primer sequences are listed in Supplementary Table S1. (Sigma-Aldrich) were dissolved in dH2O to make stock solutions at 2.5 and 6 mg/ml, respectively. ChIP assay Virus infection A ChIP assay kit (EZ-ChIP 17-371; Millipore, Billerica, MA, USA) was used Two shRNAs targeting TRIM24 were used separately in RNAi experiments according to the manufacturer’s protocol. Briefly, cells were crosslinked in for validation of results. Sense sequences were as follows: 50-AAGCAGGTG- 1% formaldehyde at room temperature for 10 min and quenched by GAACAGGATATTAAAGTTGC-3012 (shTRIM24 1) and 50-GGAATGAATCA- glycine. After washing, cells were resuspended in SDS lysis buffer. The DNA GAAGATAA-30 (shTRIM24 2). Nontargeting sense sequence was used as was sheared to fragments by sonication. The supernatant was precleared control: 50-TTCTCCGAACGTGTCACGT-30 (shControl). The pSilencer 5.1 Retro with protein G-agarose beads and then incubated with normal immu- plasmid (Ambion, Foster City, CA, USA) was used for constructing retroviral noglobulin G or anti-TRIM24 antibody overnight. After the immunopreci- vectors containing these shRNA templates. Retroviruses were produced by pitated DNA–protein crosslinks were reversed, RNA and protein transfecting pSilencer 5.1 Retro vectors into PT67 cells using Lipofectamine contamination was erased by RNase A and Proteinase K. The purified 2000 (Invitrogen, Grand Island, NY, USA). For lentivirus production, empty DNA was amplified for 30 PCR cycles using specific primers. The PCR control vector (EX-NEG-Lv105) and containing human TRIM24 ORF vector product was analyzed by 2% agarose gel electrophoresis with a DNA size (EX-T7669-Lv105) (Genecopoeia, Rochville, MD, USA) were transfected into marker. The primer sequences are listed in Supplementary Table S1. 293T cells concomitantly with packaging plasmids psPAX2 and pMD2.G (Addgene, Cambridge, MA, USA). All plasmids were confirmed by DNA Construction of expression vectors sequencing. Filtered viruses were used to infect GBM cells in the presence of 6 mg/ml polybrene. At 3 to 4 days after virus infection, puromycin The mutant TRIM24 ORF deleted of PHD–Bromo domain was PCR-amplified from the lentiviral vector using primers (forward: 50-GCTGGCTAGCAC- selection (1 mg/ml) was applied and cells without subcloning were used for 0 0 experiments. CATGGAGGTGGCGGTGG-3 and reverse: 5 -GCTTGGTACCTAGTCCTCATTG- GGGTCATCCTC-30) and cloned into the NheI and KpnI sites of the pcDNA3.1( þ ) vector (Invitrogen). For better parallel comparison, the Cell proliferation assay pcDNA 3.1( þ ) vector containing full-length human TRIM24 ORF was 0 Cells in appropriate number (5 Â 104 for T98 or 1 Â 105 for U87) were constructed by PCR amplification using primers (forward: 5 -GCTGGCTAG suspended in culture medium and plated in six-well plates per well in CACCATGGAGGTGGCGGTGG-30 and reverse: 50-GCTTGGTACCGAGCTATT triplicate. At intervals of 1 day, cells were detached with trypsinization and TAAGCAACTGGCGTTC-30). The empty vector was used as control. All the cell numbers were determined. plasmids were confirmed by DNA sequencing. Underlined are restriction sites (Nhel and Kpnl). Cell viability assay Cells in 96-well plates were exposed to different treatments. At indicated siRNA time points, MTT (Sigma-Aldrich) was added and the emerged crystal was Cells were transfected with siRNA using Lipofectamine 2000. The targeting dissolved before measurement for absorbance value. sense sequences were 50-CTCCGTGAGGCTACATTAATA-30 for PIK3CA and 50-GCTCAAGATCTGCCGAGTG-30 for RelA. Nontargeting sequence was used as control: 50-TTCTCCGAACGTGTCACGT-30. Real-time RT–PCR was Flow cytometry performed to validate gene knockdown efficiency. For cell cycle analysis, cells were harvested, fixed in 70% ethanol on ice and stained with propidium iodide in phosphate-buffered saline containing RNase. The DNA contents were analyzed by flow cytometry. For analysis of Luciferase reporter assay cell apoptosis, Annexin V–fluorescein isothiocyanate and propidium iodide Cells were co-transfected with 0.6 mgpSilencer 5.1 Retro plasmids double staining was used to sort cells in early or late apoptotic phase. containing shRNA templates, 0.18 mg pGL4.32[Luc2P/NF-kB-RE/Hygro] vector and 0.02 mg pRL-TK vector (Promega, Madison, WI, USA) using Lipofectamine 2000. Cell lysates were collected 48 h after transfection and Soft agar clone formation assay centrifuged at top speed for 1 min to clear cell debris. Luciferase activity Warmed cell culture medium containing 0.5% agar was placed into 12-well was measured using Dual-Luciferase Reporter Assay System (Promega) plates. Thereafter, 2 Â 103 cells suspended in culture medium containing and the transfection efficiency was normalized to the activity of Renilla 0.3% agar were added and the cells were fed with culture medium twice luciferase.

Oncogene (2015) 600 – 610 & 2015 Macmillan Publishers Limited TRIM24 in malignant gliomas L-H Zhang et al 609 In vivo experiments 11 Quintas-Cardama A, Qiu YH, Post SM, Zhang Y, Creighton CJ, Cortes J et al. Four-week-old female nude mice were used for xenograft studies. For T98 Reverse phase protein array profiling reveals distinct proteomic signatures cell inoculation, 5 Â 106 stably infected cells, with either shControl or associated with chronic myeloid leukemia progression and with chronic phase in shTRIM24 retroviruses, were mixed with Matrigel matrix (BD Biosciences, the CD34-positive compartment. Cancer 2012; 118: 5283–5292. San Jose, CA, USA) and the mixture was injected subcutaneously. After 12 Tsai WW, Wang Z, Yiu TT, Akdemir KC, Xia W, Winter S et al. TRIM24 6 weeks, the tumor formation status was recorded. For U87 cell links a non-canonical histone signature to breast cancer. Nature 2010; 468: inoculation, 2 Â 107 infected cells, with either shControl or shTRIM24, 927–932. were suspended in phosphate-buffered saline and directly implanted 13 Chambon M, Orsetti B, Berthe ML, Bascoul-Mollevi C, Rodriguez C, Duong V et al. subcutaneously without Matrigel matrix. After 1 week, inoculated mice Prognostic significance of TRIM24/TIF-1alpha gene expression in breast cancer. were further placed into four groups (n ¼ 6 per group): shControl þ saline, Am J Pathol 2011; 178: 1461–1469. shTRIM24 þ saline, shControl þ TMZ and shTRIM24 þ TMZ. The mice were 14 The Cancer Genome Atlas Research Network. Comprehensive genomic char- orally given 100 mg/kg/day of TMZ or equal volume of saline for 14 days.41 acterization defines human glioblastoma and core pathways. Nature 2008; At 1 week after the completion of drug treatment, the mice were killed and 455: 1061–1068. the developed tumors were weighted. 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The forkhead FOXO3a increases determine the changes in recurrent GBMs from corresponding newly phosphoinositide-3 kinase/Akt activity in drug-resistant leukemic cells through diagnosed ones. The Kaplan–Meier method was used to estimate the OS induction of PIK3CA expression. Mol Cell Biol 2008; 28: 5886–5898. and PFS curves (log-rank test). Cox proportional hazards regression models 18 Ikegami D, Akiyama H, Suzuki A, Nakamura T, Nakano T, Yoshikawa H et al. Sox9 were used to explore the prognostic values of age, sex, extent of resection, sustains chondrocyte survival and hypertrophy in part through Pik3ca-Akt Karnofsky performance score, chemotherapy status and TRIM24 expres- pathways. Development 2011; 138: 1507–1519. sion. Because of heterogeneity of variances, differences between xenograft 19 Hegi ME, Diserens AC, Gorlia T, Hamou MF, de Tribolet N, Weller M et al. MGMT weights were determined by the Kruskal–Wallis test, and the interaction gene silencing and benefit from temozolomide in glioblastoma. N Engl J Med between TRIM24 knockdown and TMZ was determined by factorial 2005; 352: 997–1003. analysis. Western blot and real-time RT–PCR data were analyzed by two- 20 Danam RP, Howell SR, Brent TP, Harris LC. Epigenetic regulation of sided Student’s t-test. All analyses were performed using the SPSS software O6-methylguanine-DNA methyltransferase gene expression by histone acetyla- package version 17.0 (SPSS, Chicago, IL, USA) and Po0.05 was considered tion and methyl-CpG binding proteins. Mol Cancer Ther 2005; 4: 61–69. to be statistically significant. 21 Tolcher AW, Gerson SL, Denis L, Geyer C, Hammond LA, Patnaik A et al. Marked inactivation of O6-alkylguanine-DNA alkyltransferase activity with protracted temozolomide schedules. Br J Cancer 2003; 88: 1004–1011. 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Supplementary Information accompanies this paper on the Oncogene website (http://www.nature.com/onc)

Oncogene (2015) 600 – 610 & 2015 Macmillan Publishers Limited