Oncogene (2014), 1–11 & 2014 Macmillan Publishers Limited All rights reserved 0950-9232/14 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 protein 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 transcription–PCR and mutation analyses demonstrate that TRIM24 binds to the PIK3CA promoter via its PHD–Bromo domain to activate the transcription of PIK3CA gene, 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 advance online publication, 27 January 2014; doi:10.1038/onc.2013.593 Keywords: glioblastoma; TRIM24; TIF1a; PI3K; Akt; MGMT

INTRODUCTION In the brain and central nervous system, 80% of malignant tumors are malignant gliomas, the majority of which are glioblastoma multiforme (GBM, World Health Organization (WHO) grade IV astrocytoma).1 Despite great advances in surgery, chemotherapy and radiotherapy, the median survival is only 12 to 15 months for 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 by a conserved N-terminal tripartite motif (TRIM) consisting of a RING domain, two B-box zinc-fingers and a coiled-coil region. In addition, its C-terminal tandem PHD finger and bromodomain distinguish the TIF subgroup from other TRIM proteins.4 TRIM24 participates in chromatin remodeling,5 targets p53 for degradation6 and interacts with nuclear receptors, either positively or negatively, modulating the transcription activity of these receptors.7 In recent years, close correlations of TRIM24 with several cancers have been unveiled. Nevertheless, its roles in cancers are seemingly controversial, such as acting as a tumor suppressor in hepatocellular carcinoma8,9 while promoting tumorigenesis in myeloid leukemia10,11 and breast cancer.12,13

In this study, we evaluated TRIM24 expression in gliomas, and addressed a positive correlation between TRIM24 level and tumor malignancy. TRIM24 promotes tumor growth and enhances resistance to chemotherapy via phosphatidylinositide 3-kinase (PI3K)/Akt signaling. Finally, possible clinical relevance of TRIM24 effect on chemotherapy response was explored.

RESULTS TRIM24 expression is positively correlated with tumor grade and participates in GBM recurrence Immunohistochemistry assay showed that normal brain glial and oligodendroglioma presented negative TRIM24 staining or weak staining in few cells. However, TRIM24 was mostly expressed in astrocytomas and was especially highly overexpressed in malignant astrocytomas that include anaplastic astrocytoma (WHO grade III) and GBM (Figure 1a). The levels of TRIM24 expression presented a tumor pathological grade-dependent pattern, except that no significant difference was observed between pilocytic astrocytoma (WHO grade I) and diffuse astrocytoma (WHO 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] 1 These two authors contributed equally to this work. Received 30 April 2013; revised 14 October 2013; accepted 18 December 2013

TRIM24 in malignant gliomas L-H Zhang et al

2 (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 expression, 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).

Figure 1. TRIM24 is overexpressed in astrocytomas, participates in GBM recurrence and is correlated with prognosis of GBM patients. (a) Immunohistochemical staining of TRIM24 in surgical samples. AA, anaplastic astrocytoma; DA, diffuse astrocytoma; NB, normal brain tissue; OG, oligodendroglioma; PA, pilocytic astrocytoma. Scale bar, 20 mm. (b) Comparison of TRIM24 levels among astrocytomas in different pathological grades. Error bars, s.d.; **Po0.01; NS, not significant. (c) Immunoblot analysis of TRIM24 protein levels in frozen preserved surgical samples. (d) Immunostaining of TRIM24 in newly diagnosed GBM and recurrent GBM samples obtained from the same patients. Shown are three representative recurrent GBM cases that presented relatively low TRIM24 expression at the first surgery. Scale bar, 20 mm. (e, f ) Kaplan–Meier estimates of prognosis in 297 newly diagnosed GBMs according to TRIM24 level. The differences in OS and PFS are both highly significant between patients with low TRIM24 expression and those with high TRIM24 expression (Po0.001 by the log-rank test).

confirmed by western blot using proteins extracted from frozen preserved tissue samples (Figure 1c). For recurrent GBM cases, compared with their first surgical samples according to which GBM was newly diagnosed, TRIM24 expression generally increased at the time of disease recurrence, especially in those who showed low expression previously Oncogene (2014) 1 – 11

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 phase, whereas forced overexpression of TRIM24 led to an increase in G2/M-phase proportion in U251 cells whose TRIM24 levels are relatively lower (Figure 2b). Reduction of TRIM24 crippled soft agar colony formation ability of T98 and U87 cells, manifested in both number and size of the clones (Figure 2c). In vivo experiments showed that once stably infected with shRNA targeting TRIM24, T98 cells failed to form subcutaneous xenograft even with the aid of Matrigel matrix, compared with the cells infected with nontargeting control shRNA (Figure 2d). To minimize individual difference among mice, T98 cells infected with either shTRIM24 or shControl were inoculated into either flank of the same mice (Figure 2e). Again, TRIM24 knockdown abrogated tumor development, further validating the growth-promoting function of TRIM24 in vivo. As activation of PI3K/Akt and Raf/MEK/extracellular-signalregulated kinase (ERK) signaling are the main pathways participating in GBM growth,14 we assessed levels of activated Akt and ERK, that represent their signaling status respectively. Knockdown of TRIM24 by targeting shRNAs suppressed phosphorylation of Akt (Figures 3a and b). Concomitantly, the phosphorylation of ERK increased, probably reflecting that the inhibition effect of activated Akt on Raf/MEK/ERK signal transduction was relieved.15 The impact of TRIM24 knockdown on cell signaling was rescued by reintroduction of TRIM24 into downregulated cells by & 2014 Macmillan Publishers Limited

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3 Table 1.

Cox proportional hazards regression analysis on newly diagnosed GBMs

Variable

Progression-free survival Univariate Cox analysis P-value

TRIM24 expression KPS Age Sex Extent of resection Chemotherapy

o0.001 o0.001 0.029 0.572 0.144 0.007

HR (95% CI) 1.50 0.98 1.01 1.07 0.87 0.72

(1.32–1.71) (0.97–0.99) (1.00–1.03) (0.84–1.36) (0.72–1.05) (0.57–0.92)

Overall survival

Multivariate Cox analysis P-value o0.001 0.001 0.093 0.201 0.074 0.095

HR (95% CI) 1.43 0.98 1.01 1.17 0.85 0.82

(1.26–1.63) (0.97–0.99) (1.00–1.02) (0.92–1.49) (0.70–1.02) (0.64–1.04)

Univariate Cox analysis P-value o0.001 o0.001 0.001 0.887 0.418 0.001

HR (95% CI) 1.60 0.98 1.02 0.98 0.93 0.67

(1.39–1.83) (0.97–0.99) (1.01–1.03) (0.77–1.25) (0.77–1.11) (0.53–0.85)

Multivariate Cox analysis P-value o0.001 0.002 0.012 0.891 0.204 0.088

HR (95% CI) 1.50 0.98 1.02 1.02 0.89 0.81

(1.30–1.72) (0.97–0.99) (1.00–1.03) (0.80–1.30) (0.75–1.07) (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.

infection with lentivirus containing human TRIM24 open reading frame (ORF), whereas empty control lentivirus had no effect (Figure 3b). Then, we hypothesized that the growth of TRIM24-downregulated cells should be less sensitive to the inhibitor of PI3K/Akt transduction because the signaling has already been suppressed therein, whereas its influence on TRIM24-overexpressed cells should be more significant because of the augmented signaling activation. This hypothesis was tested by employing LY294002, a specific inhibitor of PI3K/Akt signaling.16 Relative effect of LY294002 was evaluated, by normalizing to dimethyl sulfoxide (DMSO) that was used to dissolve LY294002. Inhibition of Akt activation had less effect on TRIM24-downregulated cells as compared with the control cells (Figure 3c). The effect of inhibition was amplified in TRIM24-overexpressed cells as compared with the cells infected with control lentivirus. In other words, inhibition of Akt phosphorylation abrogates the growth-promoting effect of & 2014 Macmillan Publishers Limited

TRIM24 (Figure 3c and Supplementary Figure S1). By the way, LY294002 induces similar effects on Akt and ERK phosphorylation as TRIM24 knockdown (Figure 3c). To evaluate whether TRIM24 regulates Akt activation in glioma tumors, immunoblot assay was employed to test the proteins in surgical samples and a significant correlation (Po0.001) was observed between TRIM24 and p-Akt levels in GBMs (Supplementary Figure S2). The levels of PI3K/Akt signaling components were then evaluated as described.17,18 Real-time reverse transcription–PCR (RT–PCR) and immunoblot analyses demonstrated the reduction of PIK3CA levels upon TRIM24 knockdown (Figures 3d and f), whereas no significant change was observed in other components. Besides, knockdown of PIK3CA using targeting small interfering RNA (siRNA) had similar effects as the pan-PI3K inhibitor LY294002 on the growth-promoting effect of TRIM24 (Supplementary Figure S3). Furthermore, chromatin immunoprecipitation (ChIP) assay demonstrated TRIM24 binding to the Oncogene (2014) 1 – 11

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4 PIK3CA promoter in T98 and U87 cells (Figure 3e). As TRIM24 recognizes histone markers by means of its PHD–Bromo domain,12 rescue experiments were performed in TRIM24-downregulated

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cells. Wild-type TRIM24 rescued PIK3CA and p-Akt levels, whereas mutant TRIM24 deleted of the PHD–Bromo domain had no effect (Figure 3f). The binding on the PIK3CA promoter was

& 2014 Macmillan Publishers Limited

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5 abrogated when the mutant TRIM24 was introduced into TRIM24downregulated cells (Figure 3g). However, there was still some sort of residual binding to the PIK3CA promoter, possibly because the elimination of wild-type TRIM24 could not be complete by RNA interference.

TRIM24 enhances resistance to temozolomide (TMZ) Chemotherapy resistance is a major cause for failed GBM treatment with alkylating agents, among which TMZ is considered 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 that TRIM24 knockdown alone had impact on cell growth, we employed the relative viability (TMZ treatment group normalized to DMSO group for either infected cells), so as to eliminate false positive confounding. MTT assay showed that downregulation of TRIM24 increased the cytotoxicity of TMZ in T98 and U87 cells (Figure 4a). Plate clone formation assay demonstrated that TMZ treatment had additive effect on TRIM24 knockdown that alone led to decreased colonies (Figure 4b). Exposure to TMZ for 1 week resulted in significant cleavage of caspase-7 in TRIM24-depleted T98 cells, whereas minimal effect was observed in the control cells (Figure 4c). TUNEL (TdT-mediated dUTP nick end labeling) and flow cytometry analyses showed that depletion of TRIM24 alone did not initiate apparent cell apoptosis. Nevertheless, apoptosis emerged evidently when TRIM24 depletion was combined with TMZ treatment (Figures 4d and e). To investigate whether TRIM24 downregulation would affect tumor chemosensitivity in vivo, U87 cells were subcutaneously inoculated into nude mice after infection with shRNAs and TMZ dissolved in saline was orally administered. Similar to the results obtained from T98 cells, which failed to form xenograft if TRIM24 was depleted, knockdown of TRIM24 in U87 cells reduced the weights of developed tumors. In addition, downregulation of TRIM24 enhanced the therapeutic effect of TMZ administration (Figure 4f) and the additive effect was significant (Po0.01), as determined by factorial analysis. TMZ-induced injury can be repaired by O6-methylguanine-DNA methyltransferase (MGMT), a DNA repair enzyme whose key role in TMZ resistance is now commonly recognized.19 Thus, we determined possible change in MGMT expression level after TRIM24 knockdown. After infection with shRNAs targeting TRIM24, the cells expressed less MGMT as compared with parental cells and cells infected with shControl (Figure 5a). Real-time RT–PCR analysis demonstrated that TRIM24 knockdown reduced MGMT in the transcriptional level (Figure 5b). However, we did not observe TRIM24 binding to the MGMT promoter by employing ChIP assay (data not shown), using primers described previously.20 Nevertheless, TRIM24 regulates PI3K/Akt signaling that activates nuclear factor (NF)-kB, and NF-kB has binding sites within the

MGMT promoter. To test this hypothesis, we employed a luciferase reporter construct containing NF-kB response elements and found that NF-kB activity was reduced upon TRIM24 knockdown (Figure 5c). Similar results were observed when cells were treated with LY294002 (data not shown). Moreover, the effect of TRIM24 knockdown on MGMT expression was rescued by reintroduction of TRIM24 into downregulated cells by infection with lentivirus containing TRIM24 ORF, whereas empty control lentivirus had no effect. Transfection of siRNA targeting RelA, which encodes a subunit of NF-kB, abolished the rescue effect of TRIM24 (Figure 5d). Because MGMT consumption takes place during the DNA repair process,21 we hypothesized that apart from affecting baseline level of MGMT, TRIM24-depleted cells might fail to replenish enough MGMT to counteract insistent TMZ exposure, and this consequence could appear more pronounced as the intensity of TMZ action escalated. Our hypothesis was tested by exposing cells to TMZ at increased concentrations for 1 week, a period enough for the consumption of preliminarily preserved MGMT. As expected, the decrease in MGMT levels presented a TMZ dosedependent manner in TRIM24-depleted cells, with the lowest MGMT level in the highest 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 itself in the plate clone formation assay (Figure 4b). Besides, significant correlation (Po0.01) between TRIM24 and MGMT levels was observed in GBMs (Supplementary Figure S4). Low TRIM24 expression is a prerequisite for effective chemotherapy In light of TRIM24 effect on chemosensitivity in lab assays and nearly half of GBM patients in this study did not receive chemotherapy, we thus attempted to explore the impact of TRIM24 level on chemotherapy in the clinical context. To this end, GBM patients were subdivided according to TRIM24 expression level in their tumors and the therapeutic effect of chemotherapy was separately analyzed. Interestingly, a significant impact of TRIM24 on chemotherapy response was noticed. That is, in the low TRIM24 expression subgroup, patients who received chemotherapy had a median OS of 14.8 months as compared with 9.5 months for those who did not receive chemotherapy (P ¼ 0.008; Figure 6a). Moreover, in this subgroup, those who received chemotherapy had a median PFS of 6.6 months as compared with 5.1 months for those who did not receive chemotherapy (P ¼ 0.042; Figure 6b). On the other hand, among the patients whose tumors displayed high TRIM24 expression, no significant prognostic benefit from chemotherapy was observed (Figures 6c and d). Besides, the multivariate Cox regression analysis confirmed the independent role of TRIM24 expression in chemotherapy response.

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 gene expression 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. & 2014 Macmillan Publishers Limited

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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 Patients suffering from malignant gliomas display heterogeneous clinical outcome, especially among GBM patients, although their tumors appear histologically similar.22 Identification of tumor biomarkers can aid in prognostic prediction and modulation of conventional therapies, as well as molecular targeting approaches. In this study, we discovered that the expression of TRIM24 not only presented a tumor grade-dependent pattern but also distinguished patients with good prognosis from those with poor outcome within a single grade-GBM, suggesting that TRIM24 detection may improve the predictive strength of currently available prognostic indicators. However, we could not address the relationship of TRIM24 expression and prognosis of patients with gliomas in other pathological grades because of not enough prognostic information about them. The upregulation of TRIM24 at GBM recurrence might imply the contribution of evolutional pressure, because overexpressed cells may possess greater proliferation rate and circumvent conventional therapeutic Oncogene (2014) 1 – 11

approaches, whereas the cells with low expression are gradually replaced or eradicated by treatments. The effects of TRIM24 on GBMs manifest in tumor growth and chemoresistance. The growth-promoting effect of TRIM24 emanates through PI3K/Akt signaling pathway that is often overactivated in GBMs to stimulate cell proliferation and inhibit apoptosis.23 TRIM24 also promotes breast cancer development as a potent coactivator of estrogen receptor-a,12 and it also enhances prostate cancer progression by activating androgen receptor.24 On the other hand, TRIM24 suppresses hepatocarcinogenesis by inactivation of retinoic acid receptor-a8,25 that affects interferon/ signal transducer and activator of transcription (IFN/STAT) pathway.26,27 TRIM24 also prevents arterial calcification by suppressing the vitamin D receptor signaling pathway.28 These conflicting findings might be because of the switch of TRIM24 roles between activator and suppressor according to different cellular pathways, and what signaling is pivotal in a specific disease, thus ultimately dictating in which direction the balance & 2014 Macmillan Publishers Limited

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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 induces proliferation of normal hepatocyte,29 whereas adaptive mechanism that is activated to fight against hyperproliferation can lead to increased ploidy that is prone to initiate carcinogenesis in the liver.26 However, activation of retinoic acid receptor-a in GBMs indicates longer survival for patients.30 In addition, TRIM24 has been found to be a negative regulator of the tumor suppressor p53 in embryonic stem cells and several cancer cell lines,6,31 but the p53 regulation mechanism may be different in liver. For instance, irradiation cannot induce p53 accumulation in the liver,7 and MDM2 loss does not alleviate the instability of mutant p53 in the liver, whereas it does so in the brain.32 Nevertheless, TRIM24 seems to have p53-independent effect in gliomas, because U87 cells express wild-type p53 whereas T98 and U251 cells express mutant p53, similar to the results obtained from lung cancer.33 Knockdown of TRIM24 in T98 and U87 cells increased the levels of p53 protein (Supplementary Figure S5). However, the baseline level and induced level of p53 were both higher in T98 cells than those in U87 cells, owing to its mutant form in the former.34 Therefore, the different sensitivity to TMZ-induced cytotoxicity in & 2014 Macmillan Publishers Limited

T98 and U87 cells might be attributed to their p53 status, TRIM24 knockdown efficiency and, of course, MGMT expression profile.35 TRIM24 binds to the PIK3CA promoter via its PHD–Bromo domain and induces the overexpression of PIK3CA that in turn augments the phosphorylation of Akt. Phosphorylated Akt activates MDM236 that promotes p53 degradation.37 Therefore, besides direct interaction with p53, TRIM24 might indirectly regulate p53 level through PI3K/Akt signaling. As Ras activates PI3K38 whereas p-Akt inhibits Raf,36 the increased phosphorylation of ERK could reflect that the inhibition effect of activated Akt on Raf/MEK/ERK signal transduction was relieved. In addition, considering that TRIM24 knockdown inhibits cell proliferation, the changes in ERK activation are more likely to be secondary and compensatory, indirectly demonstrating that TRIM24 acts on PI3K/ Akt signaling via regulating PIK3CA expression (Figure 7). Apart from its role in tumor growth, TRIM24 enhances resistance to alkylating chemotherapy by regulating the DNA repair enzyme MGMT that removes alkyl groups from the O6 position of guanine and blunts the cytotoxicity induced by chemotherapy.39 Our results suggest that TRIM24 regulates MGMT Oncogene (2014) 1 – 11

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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 Figure 7. Schematic diagram depicting TRIM24-regulated pathways in glioma cells. TRIM24 activates PI3K/Akt signaling that subsequently elevates NF-kB activity. Activated NF-kB translocates into the nucleus and binds to the MGMT promoter, thus enhancing its transcription. Upregulation of the PI3K/Akt pathway promotes cell proliferation and inhibits apoptosis induced by chemotherapy, 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 MDM2.

expression through PI3K/Akt/NF-kB signaling transduction. As TRIM24 is overexpressed in GBMs, especially those leading to poorer prognosis, TRIM24 may be considered as an attractive therapeutic target alone or in combination with conventional Oncogene (2014) 1 – 11

Surgical samples were collected at the Department of Neurosurgery, Xijing Hospital between 2005 and 2010. They included normal brain tissue (n ¼ 15), pilocytic astrocytoma (WHO grade I; n ¼ 24), diffuse astrocytoma (WHO grade II; n ¼ 57), anaplastic astrocytoma (WHO grade III; n ¼ 63), newly diagnosed GBM (WHO grade IV; n ¼ 297), recurrent GBM (n ¼ 48) and oligodendroglioma (n ¼ 44).40 All patients provided written informed consent and this study was approved by the Institutional Review Board.

Immunohistochemistry Formalin-fixed, paraffin-embedded tissues were employed for immunohistochemistry with anti-TRIM24 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and nuclei were slightly counterstained with hematoxylin. The intensity and percentage of positive cells were evaluated in at least five separate fields at  400 magnification. The scores were evaluated by two pathologists who were blinded to clinical data. Immunoreactivity was scored as follows: 0, no staining; 1, weak staining & 2014 Macmillan Publishers Limited

TRIM24 in malignant gliomas L-H Zhang et al

9 in o50% cells; 2, weak staining in X50% cells; 3, strong staining in o50% cells; and 4, strong staining in X50% cells.

per week. After 2 weeks, colonies larger than 50 mm in diameter were recorded in five separate fields.

Western blot

Plate clone formation assay

Tissue and cell samples were lysed in RIPA buffer supplemented with 1 mM NaF, 1 mM Na3VO4 and protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany). Equal amounts of total protein were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. The primary antibodies against TRIM24, PIK3CA, Actin (Santa Cruz Biotechnology), p-Akt (Ser 473), Akt, p-ERK1/2, ERK1/2, procaspase-7, cleaved caspase-7 (Cell Signaling Technology, Beverly, MA, USA), MGMT, p53 (Bioworld Technology, St Louis, MO, USA) were used according to the manufacturers’ recommendations. Each immunoblot was done at least thrice and the signals were quantified using ImageJ software (Bethesda, MD, USA).

For clone formation, 150 cells were seeded in 60 mm dishes after infection with shRNAs. After cell attachment, cultures were exposed to TMZ in different concentrations. After 2 weeks, cells were stained with 0.25% crystal violet and the colonies containing more than 50 cells were recorded.

Cell culture and drugs 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 bovine serum at 37 1C in 5% CO2. TMZ (Sigma-Aldrich, St Louis, MO, USA) was dissolved in DMSO (Sigma-Aldrich) at a stock concentration of 100 mmol/l and stored at  20 1C. For in vivo studies, TMZ (ScheringPlough, Kenilworth, NJ, USA) was dissolved in saline (4 mg/ml) for oral administration. A 10 mM solution of LY294002 in DMSO was purchased from Merck Biosciences (Darmstadt, Germany). Puromycin and polybrene (Sigma-Aldrich) were dissolved in dH2O to make stock solutions at 2.5 and 6 mg/ml, respectively.

TUNEL assay After treatment, cells grown in the glass bottom dishes were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. The DNA 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 confocal fluorescent microscope (Olympus, Shinjuku, Tokyo, Japan).

Real-time RT–PCR Total RNA was isolated with RNAiso Plus (Takara, Dalian, China) and reverse transcribed with PrimeScript RT Master Mix (Takara). PCR amplification was performed in triplicate with SYBR Premix Ex Taq II (Takara) using CFX96 Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). The values were normalized to the levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Part of the primer sequences are listed in Supplementary Table S1.

ChIP assay Virus infection Two shRNAs targeting TRIM24 were used separately in RNAi experiments for validation of results. Sense sequences were as follows: 50 -AAGCAGGTGGAACAGGATATTAAAGTTGC-30 12 (shTRIM24 1) and 50 -GGAATGAATCAGAAGATAA-30 (shTRIM24 2). Nontargeting sense sequence was used as control: 50 -TTCTCCGAACGTGTCACGT-30 (shControl). The pSilencer 5.1 Retro plasmid (Ambion, Foster City, CA, USA) was used for constructing retroviral vectors containing these shRNA templates. Retroviruses were produced by transfecting pSilencer 5.1 Retro vectors into PT67 cells using Lipofectamine 2000 (Invitrogen, Grand Island, NY, USA). For lentivirus production, empty control vector (EX-NEG-Lv105) and containing human TRIM24 ORF vector (EX-T7669-Lv105) (Genecopoeia, Rochville, MD, USA) were transfected into 293T cells concomitantly with packaging plasmids psPAX2 and pMD2.G (Addgene, Cambridge, MA, USA). All plasmids were confirmed by DNA 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 selection (1 mg/ml) was applied and cells without subcloning were used for experiments.

Cell proliferation assay Cells in appropriate number (5  104 for T98 or 1  105 for U87) were suspended in culture medium and plated in six-well plates per well in triplicate. At intervals of 1 day, cells were detached with trypsinization and the cell numbers were determined.

Cell viability assay Cells in 96-well plates were exposed to different treatments. At indicated time points, MTT (Sigma-Aldrich) was added and the emerged crystal was dissolved before measurement for absorbance value.

Flow cytometry 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 cell apoptosis, Annexin V–fluorescein isothiocyanate and propidium iodide double staining was used to sort cells in early or late apoptotic phase.

Soft agar clone formation assay Warmed cell culture medium containing 0.5% agar was placed into 12-well plates. Thereafter, 2  103 cells suspended in culture medium containing 0.3% agar were added and the cells were fed with culture medium twice & 2014 Macmillan Publishers Limited

A ChIP assay kit (EZ-ChIP 17-371; Millipore, Billerica, MA, USA) was used according to the manufacturer’s protocol. Briefly, cells were crosslinked in 1% formaldehyde at room temperature for 10 min and quenched by glycine. After washing, cells were resuspended in SDS lysis buffer. The DNA was sheared to fragments by sonication. The supernatant was precleared with protein G-agarose beads and then incubated with normal immunoglobulin G or anti-TRIM24 antibody overnight. After the immunoprecipitated DNA–protein crosslinks were reversed, RNA and protein contamination was erased by RNase A and Proteinase K. The purified DNA was amplified for 30 PCR cycles using specific primers. The PCR product was analyzed by 2% agarose gel electrophoresis with a DNA size marker. The primer sequences are listed in Supplementary Table S1.

Construction of expression vectors The mutant TRIM24 ORF deleted of PHD–Bromo domain was PCR-amplified from the lentiviral vector using primers (forward: 50 -GCTGGCTAGCACCATGGAGGTGGCGGTGG-30 and reverse: 50 -GCTTGGTACCTAGTCCTCATTGGGGTCATCCTC-30 ) and cloned into the NheI and KpnI sites of the pcDNA3.1( þ ) vector (Invitrogen). For better parallel comparison, the pcDNA 3.1( þ ) vector containing full-length human TRIM24 ORF was constructed by PCR amplification using primers (forward: 50 -GCTGGCTAG CACCATGGAGGTGGCGGTGG-30 and reverse: 50 -GCTTGGTACCGAGCTATT TAAGCAACTGGCGTTC-30 ). The empty vector was used as control. All plasmids were confirmed by DNA sequencing. Underlined are restriction sites (Nhel and Kpnl).

siRNA Cells were transfected with siRNA using Lipofectamine 2000. The targeting 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 performed to validate gene knockdown efficiency.

Luciferase reporter assay Cells were co-transfected with 0.6 mg pSilencer 5.1 Retro plasmids 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 centrifuged at top speed for 1 min to clear cell debris. Luciferase activity was measured using Dual-Luciferase Reporter Assay System (Promega) and the transfection efficiency was normalized to the activity of Renilla luciferase. Oncogene (2014) 1 – 11

TRIM24 in malignant gliomas L-H Zhang et al

10 In vivo experiments Four-week-old female nude mice were used for xenograft studies. For T98 cell inoculation, 5  106 stably infected cells, with either shControl or shTRIM24 retroviruses, were mixed with Matrigel matrix (BD Biosciences, San Jose, CA, USA) and the mixture was injected subcutaneously. After 6 weeks, the tumor formation status was recorded. For U87 cell inoculation, 2  107 infected cells, with either shControl or shTRIM24, were suspended in phosphate-buffered saline and directly implanted subcutaneously without Matrigel matrix. After 1 week, inoculated mice were further placed into four groups (n ¼ 6 per group): shControl þ saline, shTRIM24 þ saline, shControl þ TMZ and shTRIM24 þ TMZ. The mice were orally given 100 mg/kg/day of TMZ or equal volume of saline for 14 days.41 At 1 week after the completion of drug treatment, the mice were killed and the developed tumors were weighted. Animal experiments were approved by the Institutional Animal Care and Use Committee, and all procedures were conducted in accordance with the institutional guidelines.

Statistical analysis Because of nonparametric distribution of immunohistochemical scores, the Kruskal–Wallis test was used to compare astrocytomas between every two pathological grades, whereas the Wilcoxon signed-rank test was used to determine the changes in recurrent GBMs from corresponding newly diagnosed ones. The Kaplan–Meier method was used to estimate the OS and PFS curves (log-rank test). Cox proportional hazards regression models were used to explore the prognostic values of age, sex, extent of resection, Karnofsky performance score, chemotherapy status and TRIM24 expression. Because of heterogeneity of variances, differences between xenograft weights were determined by the Kruskal–Wallis test, and the interaction between TRIM24 knockdown and TMZ was determined by factorial analysis. Western blot and real-time RT–PCR data were analyzed by twosided Student’s t-test. All analyses were performed using the SPSS software package version 17.0 (SPSS, Chicago, IL, USA) and Po0.05 was considered to be statistically significant.

CONFLICT OF INTEREST The authors declare no conflict of interest.

ACKNOWLEDGEMENTS We thank Xiao-Liang Yang, Xi-Qiang Cai and Juan Li for helpful assistance. This work was supported by the National Natural Science Foundation of China (no. 30870843 and no. 81171087) to X Zhang. The funding body does not play any role in the design of the study, collection and analysis of data and decision to publish.

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Supplementary Information accompanies this paper on the Oncogene website (http://www.nature.com/onc)

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