IDH1-R132H Suppresses Glioblastoma Malignancy through FAT1-ROS-HIF-1α Signaling
Correspondence Address: Source of Support: None, Conflict of Interest: None DOI: 10.4103/0028-3886.294557
Source of Support: None, Conflict of Interest: None
Keywords: Glioblastoma, HIF-1α, FAT1, IDH1-R132H, ROS
Glioblastomas (GBM) is an aggressive type of tumor that can occur in the brain or spinal cord, which has a poor survival rate and high tumor heterogeneity. Despite the advanced therapeutic strategy, including surgery, radiotherapy, and chemotherapy, the median survival of GBM patients remains ∼15 months., The mutation of the genome is closely associated with the outcome of GBM. Genetic changes resulted from mutation can influence cell survival and death pathways, leading to dysregulation of cell fate.
Isocitrate dehydrogenase 1 (IDH1) is the enzyme responsible for the conversion of isocitrate to alpha-ketoglutarate (α-KG), generating nicotine adenine disphospho nucleotide (NADPH) in the cytoplasm and peroxisomes. The mutation of IDH1 was found, in 2008, in more than 70% of primary gliomas and secondary GBM., IDH mutation predicts a better prognosis in GBM patients than those with IDH wild-type (IDH1-WT). R132H mutation accounts for more than 90% of the total mutations in IDH1. It has been identified that R132H mutation of IDH1 is an early event in the development of glioma, preceding TP53 mutations. IDH1 functions to regulate a variety of cellular functions including redox modulation, glucose sensing, and lipogenesis. However, the mechanism of IDH1-R132H-induced effect on GBM is still not clear. IDH1-R132H is associated with increased hypoxia-inducible factor-1α (HIF-1α) expression in GBM tumors. HIF-1α and its downstream targets are expressed in gliomas and estimated to predict the outcomes of GBM patients. However, the interaction of IDH1-R132H and HIF-1α in the development of GBM needs to be further exploration.
The present study was designed to explore the mechanism of IDH1-R132H-induced regulation of HIF-1α in GBM. We identified that IDH1-R132H could increase the expression of FAT Atypical Cadherin 1 (FAT1), promote the generation of reactive oxygen species (ROS), and then result in the upregulation of HIF-1α, finally leading to decreased proliferation and increased apoptosis.
Cell lines and cell culture
Human U87 cells were purchased from American Type Culture Collection (Manassas, VA, USA). U251 GBM cell lines were ordered from the Shanghai Institute of Biochemistry and Cell Biology (Shanghai, China). All the cells were cultured and maintained in DMEM (Gibco-BRL, NY, USA) supplemented with 10% fetal bovine serum (FBS) (Invitrogen, Carlsbad, CA, USA), 100 U/mL penicillin, and 100 mg/mL streptomycin and incubated at 37°C with a 5% CO2 humidified atmosphere.
The overexpression of IDH1-WT and IDH1-R132H was performed as previously described (Cui et al., 2016). In brief, to construct IDH1-WT, we sub-cloned the coding fragment into the pLenti6.3-MCS-IRES2 vector (Invitrogen, USA) using the restriction sites for BamHI and AscI. The primer sequences were as follows: forward primer 5'-ATGTCCAAAAAAATCAGTGGCGG-3' and reverse primer 5'-GTTTGGCCTGAGCTAGTTTG-3'. To construct IDH1-R132H, we sub-cloned mutated form of IDH1 at Arg 132 into the vector. An empty vector was used as the control. Lentivirus carrying shHIF-1α, shFAT1 and their negative control (shNC) were constructed by Genepharma company (Shanghai, China). In brief, cells were plated onto 35mm dishes. Fresh DMEM without FBS was added when cells were at 80% confluence. After that, 10 μL lentivirus solutions were added into the medium and incubated for an additional 24 h. To select the transfected cells, 2 μM puromycin (Sigma, Shanghai, China) was added into the culture medium 3 days after the transfection and then cells were cultured for 24 h.
Cell proliferation was evaluated by the use of MTT assay kit (Beyotime, Shanghai, China) according to the manufacturer's protocols. In brief, at indicated experimental time, cells were incubated with serum-free culture medium containing 20% MTT solutionat 37°C for 4 h. The absorbance at 1, 2, 3, 4, and 5 days was measured using a microplate reader (Bio-Rad, CA, USA).
Flow cytometric analysis of apoptosis and ROS level
TUNEL Apoptosis Detection Kit (Roche, Switzerland) was used to evaluate apoptosis. TUNEL reaction solution was added into cell suspension and incubated at 37°C for 1 h according to the manufacturer's instructions. After washing, the percentage of TUNEL-positive cells were analyzed using flow cytometry. Relative apoptosis was expressed as fold change versus control. DCFH-DA oxidant sensitive probe was used to detect ROS level. Cell suspension (106/ml) was incubated with 10 μM DCFH-DA in serum-free medium for 1h at 37°C. After washing with PBS, fluorescence intensity was analyzed using flow cytometry.
Invasion and migration assays
Cell migration ability was evaluated using wound healing assay. Cells were plated onto 6-well plates. When cell confluence reached 100%, 200-μl sterile pipette tip was used to make a linear scratch. Cells were allowed to grow in serum-free medium for an additional 24 h. The distance of scratch was captured using a microscope. The relative recovery distance of the scratch was expressed as fold change versus control. Invasion ability of GBM cells was evaluated using transwell assay. Cells were seeded into the upper chamber coated with Matrigel which was inserted into a 24-well plate. The upper chamber contained serum-free medium and the lower chamber was supplemented with medium containing 10% FBS. After 24 h incubation, cells that adhered to the lower well were fixed, stained and counted. Relative numbers of invaded cells were expressed as fold change versus control.
Xenograft tumor model in mice
All animal experiments were approved by the Institutional Animal Care and Use Committee of the Fifth Affiliated Hospital of Zhengzhou University and in accordance with ARRIVE and NIH guidelines for animal welfare. 1 × 107 U87 cells stably expressing either wild type IDH1 or IDH1-R132H and transfected with shFAT1 or shNC, were injected subcutaneously into the flanks of 5-week-old nude male BALB/cA-nu mice (Mode Animal Institute, Nanjing, China; n = 6 per group). Tumor growth was measured at 7, 14, 21, 28, and 35 days with a digital caliper. Tumor volumes were calculated as 0.5 × length × width2 using caliper. After sacrifice of the mice, tumor weight was calculated.
RNA extraction and quantitative real-time polymerase chain reaction
TRIzol reagent (Life Technologies, Carlsbad, CA, USA) was used to extract total RNA and isolated RNA was quantified using a NanoDrop ND-2000. RNA was used to reversely transcribe into cDNA by the use of RT reagent Kit with gDNA Eraser (Takara, Dalian, China) according to the manufacturer's instructions. Quantitative RT-PCR (RT-qPCR) was performed using SYBR Green Master Mix (Takara, Dalian, China) on a Bio-Rad system. GAPDH was used as the internal control. PCR reaction conditions were as follows: 94°C for 30 seconds and 40 cycles of amplification (94°C for 5 seconds, 60°C for 30 seconds and 72°C for 30 seconds). Relative level of mRNA expression was calculated using the 2− ΔΔCt method.
Western blotting analysis
At the end of the experiments, cells in 6-well plates were washed with cold PBS and then lysed in 150 μL of RIPA lysis buffer containing protease inhibitor. Protein concentration was measured using BCA assay (Thermo Fisher Pierce, Rockford, USA). Lysates were mixed with equal volume of SDS loading buffer. 20 μg of protein mixture was loaded in each lane and resolved using SDS-PAGE gel electrophoresis. Then, protein was transferred onto a PVDF membrane (Millipore, Billerica, MA, USA). Blocking was performed using 5% skimmed milk for 1 h at room temperature. After that, membranes were probed with specific antibodies, including IDH1 (1:1000, CST, Shanghai, China), IDH1-R132H (1:1000, Sigma, Shanghai, China), FAT1 (1:1000, Santa Cruz, Dallas, USA) and HIF-1α (1:1000, CST, Shanghai, China). β-actin primary antibody (1:10000, Thermo Fisher Pierce, MA, USA) was used as the internal control. Horseradish peroxidase (HRP)-conjugated secondary antibodies were used to detect the primary antibodies. Immunoblot bands were visualized using the enhanced chemiluminescence (ECL) system (Thermo Fisher Pierce, Rockford, IL, USA).
Experiments were performed in triplicate. Data were showed as means ± standard deviation (SD) and analyzed using Graph pad Prism software 6.0. Statistical analysis was performed using one-way analysis of variance followed by Student-Newman-Keuls test. The P value <0.05 was considered to be statistically significant.
IDH1-R132H suppresses proliferation, migration, invasion and increases apoptosis in glioblastoma cells
Firstly, we tested the effect of R132H mutation of IDH1 on malignancy in GBM cells, using U87 and U251 cells. U87 and U251 cells were transfected with IDH1-WT or IDH1-R132H and the effect of IDH1-R132H on proliferation, apoptosis, migration, and invasion was determined. The efficiency of IDH1-R132H transfection was confirmed by western blot detection of IDH1-R132H protein expression [Figure 1]a. In both U87 and U251 cells, IDH1-R132H significantly decreased the cell proliferation [Figure 1]b and [Figure 1]c. In contrast, cell proliferation was not affected by the transfection of IDH1-WT [Figure 1]b and [Figure 1]c. Alteration of apoptosis was also evaluated. In [Figure 1]d and e], we showed that IDH1-R132H, but not IDH1-WT, notably increased apoptosis in U87 and U251 cells. Additionally, IDH1-R132H markedly decreased the relative migration and invasion of U87 and U251 cells [Figure 1]f, [Figure 1]G, [Figure 1]H, [Figure 1]i. In contrast, the ability of migration and invasion was not affected by IDH1-WT in U87 and U251 cells [Figure 1]f, [Figure 1]g, [Figure 1]h, [Figure 1]i. In combination with previous studies, the results demonstrated that IDH1-R132H played an inhibitory role in GBM cell proliferation.
IDH1-R132H enhances TMZ toxicity in glioblastoma cells
Temozolomide (TMZ), an alkylating agent, is the first choice of chemotherapeutic drugs for glioma treatment, which can cause DNA lesions and cell apoptosis through inducing methylation on DNA bases in several positions. In the current study, we also tested the effect of IDH1-R132H on TMZ-induced cytotoxicity. As shown in [Figure 2]a and [Figure 2]b, TMZ significantly decreased cell proliferation, compared with non-treated controls, in both U87 and U251 cells. IDH1-R132H over expression markedly promoted TMZ-induced decrease of cell proliferation [Figure 2]a and [Figure 2]b. In addition, TMZ significantly increased apoptosis, compared with non-treated controls, in both U87 and U251 cells [Figure 2]c and [Figure 2]d. IDH1-R132H overexpression markedly promoted TMZ-induced increase of apoptosis [Figure 2]c and [Figure 2]d. The results demonstrated that IDH1-R132H enhances TMZ-induced cytotoxicity in glioblastoma cells.
Increased expression of HIF-1α is involved in IDH1-R132H-induced inhibition of glioblastoma malignancy
Previous studies have found that IDH1-R132H is associated with increased HIF-1α expression in GBM tumors. To explore the possible role of HIF-1α in IDH1-R132H-induced effect on GBM cell malignancy, expression of HIF-1α was determined after the overexpression of IDH1-R132H. As shown in [Figure 3]a, overexpression of IDH1-R132H significantly increased the protein expression of HIF-1α in both U87 and U251 cells. Moreover, IDH1-R132H over expression resulted in a significant increase of HIF-1α mRNA expression in both U87 and U251 cells [Figure 3]b and [Figure 3]c. In contrast, IDH1-WT over expression had no significant effect on HIF-1α expression [Figure 3]a, [Figure 3]b, [Figure 3]c. To further testify the role of HIF-1α upregulation, U87 and U251 cells were transfected with LV-shHIF-1α to downregulate the expression of HIF-1α [Figure 3]d. As shown in [Figure 3]e and [Figure 3]f, downregulation of HIF-1α expression significantly suppressed IDH1-R132H-induced decrease of cell proliferation. In addition, decrease of HIF-1α expression inhibited IDH1-R132H-induced increase of apoptosis [Figure 3]g and [Figure 3]h. IDH1-R132H-resulted enhancement of cytotoxicity induced by TMZ was also inhibited by downregulation of HIF-1α expression [Figure 3]g and [Figure 3]h. The results demonstrated that upregulation of HIF-1α is involved in IDH1-R132H-induced inhibition of glioblastoma malignancy.
Generation of ROS is involved in IDH1-R132H-induced increase of HIF-1αexpression in glioblastoma cells
It has been reported that IDH1 mutation can result in the decline of NADPH and induce reactive oxygen species (ROS) accumulation. In the present study, we examined effect of IDH1-R132H overexpression on ROS level in U87 and U251 cells. As shown in [Figure 4]a, overexpression of IDH1-R132H induced a significant increase of ROS level in both U87 and U251 cells. Overexpression of IDH1-R132H also resulted in a reduction of antioxidant enzyme expression, including superoxide dismutase 2 (SOD2) and glutamate-cysteine ligase catalytic subunit (GCLC) [Figure 4]b. To explore the role of ROS generation in the regulation of HIF-1α expression, U87 and U251 cells stably expressing IDH1-R132H were treated by N-Acetylcysteine (NAC), an antioxidant. We showed that NAC treatment significantly inhibited IDH1-R132H-induced increase of HIF-1α expression [Figure 4]c. These findings suggested that ROS generation was involved in IDH1-R132H-induced regulation of HIF-1α.
Increased expression of FAT1 is involved in IDH1-R132H-induced inhibition of glioblastoma malignancy
It has been reported previously that FAT1 was an upstream regulator of HIF-1α. Whether FAT1 was involved in IDH1-R132H-induced regulation of HIF-1α was not known. In this study, we found that overexpression of IDH1-R132H significantly increased the protein expression of FAT1 in both U87 and U251 cells. In contrast, IDH1-WT overexpression had no significant effect on FAT1 expression [Figure 5]a. To further testify the role of FAT1 upregulation, U87 and U251 cells were transfected with LV-shFAT1 to downregulate the expression of FAT1 [Figure 5]b. As shown in [Figure 5]c, downregulation of FAT1 expression significantly suppressed IDH1-R132H-induced decrease of cell proliferation. In addition, decrease of FAT1 expression inhibited IDH1-R132H-induced increase of apoptosis [Figure 5]d. IDH1-R132H-resulted enhancement of cytotoxicity induced by TMZ was also inhibited by downregulation of FAT1 expression [Figure 5]d. Furthermore, downregulation of FAT1 significantly inhibited IDH1-R132H-induced generation of ROS [Figure 5]e. The results demonstrated that increased expression of FAT1 was involved in IDH1-R132H-induced inhibition of glioblastoma malignancy through regulation of ROS generationandHIF-1α expression.
IDH1-R132H increasedthe expression of FAT1 and inhibited glioblastoma growth in implanted mice in vivo
The inhibitory effect of IDH1-R132H and FAT1 on GBM was further verified in xenograft mice in vivo. As shown in [Figure 6]a, overexpression of IDH1-R132H induced a significant decrease of implanted tumor growth. The tumor weight and tumor volume were reduced by overexpression of IDH1-R132H [Figure 6]b and [Figure 6]c. In contrast, the downregulation of FAT1 remarkably inhibited IDH1-R132H-induced inhibition of tumor growthin vivo[Figure 6]a, [Figure 6]b, [Figure 6]c. These results further confirmed the inhibitory role of IDH1-R132H-FAT1 pathway in GBM development.
R132H is a common form of mutation in IDH gene in GBM patients and predicts a better prognosis than those patients with IDH1-WT. However, the exact role of IDH1-R132H in GBM is still controversial. The molecular mechanism underlying the beneficial effect of R132H mutation of IDH1 is still far from completely understood. Nie et al. found thatIDH1-R132H decreased the proliferation of U87 cells through the upregulation of microRNA-128a. However, the same group also found that IDH1-R132H enhanced the proliferation of A 172 cells via regulation of aerobic glycolysis. In addition, R132H mutation in IDH1 gene inhibits proliferation, cell survival and invasion of glioma via downregulation of Wnt/β-catenin signaling. Moreover, R132H mutation of IDH is associated with increased expression of HIF-1α and its downstream targets. Miroshnikova et al. identified that IDH1 mutation restricted glioma aggression by reducing HIF-1α-dependent tenascin C. In contrast, Wang et al. reported that R132H mutation of IDH1 induced glioma cell proliferation via activation of nuclear factor-kappa B in a HIF-1α-dependent manner.
In the present study, we confirmed the inhibitory role of IDH1-R132H in GBM, as evidenced by the suppressive effect of IDH1-R132H overexpression on proliferation, invasion, migrationin vitro and tumor growthin vivo and the promotive effect of IDH1-R132H overexpression on apoptosis and chemosensitivity to TMZ. In combination with previous reports, most of the studies suggest that IDH1-R132H plays an inhibitory role in GBM survival. Contradictory results may be attributed to the use of different cell types and different status of cell lines.
We further explored that HIF-1α was a downstream target that mediated the inhibitory role of IDH1-R132H in GBM. Under numerous pathological conditions, HIF-1αexpression could be regulated through transcriptional and post translational modulation. In our study, we showed that mRNA expression of HIF-1α was increased by IDH1-R132H, indicating that IDH1-R132H inhibits GBM malignancy through transcriptional activation of HIF-1α. Considering the reported role of HIF-1α in the regulation of angiogenesis in various types of tumor, we also evaluated angiogenesis. However, we did not observe any significant effect of IDH1-R132H on angiogenesis under the present condition (data not shown). A recent report showed that IDH1 mutation was associated with lower expression of VEGF but not microvessel formation in GBM. The results suggested that abnormal angiogenesis may not be involved in the IDH1-R132H-induced regulation of GBM malignancy.
IDH1 dysfunction could induce energy metabolism disorders and cause the accumulation of ROS in cells. Furthermore, many studies also reported that the ROS level in IDH1-R132H cells was increased. HIF-1α is a ROS sensitive transcription factor., In this study, we showed that IDH1-R132H overexpression induced a significant increase of ROS level. The treatment of antioxidant NAC notably inhibited IDH1-R132H overexpression-induced upregulation of HIF-1α expression. The results suggest that ROS was a mediator of IDH1-R132H-induced regulation of HIF-1α. However, in the current study, the mechanism of ROS-mediated transcriptional upregulation of HIF-1α was still not clarified. There may be other redox-sensitive transcription factors that regulate the expression of HIF-1αin response to ROS.
In recent years, several studies have found that FAT1 was an upstream regulator in the regulation of gliomas., We also examined the possible role of FAT1 in IDH1-R132H-induced regulation of HIF-1α. We found that overexpression of IDH1-R132H upregulated FAT1 and downregulation of FAT1 decreased HIF-1α expression. Additionally, downregulation of FAT1 also inhibited overexpression of IDH1-R132H-induced decrease of GBM cell survival and ROS generation, and tumor growth in xenograft mice. The results provide the evidence for the role of FAT1 in IDH1-R132H-induced ROS generation, increase of HIF-1α expression and decrease of cell survival in GBM cells. However, our study did not elucidate the molecular mechanism underlying IDH1-R132H-induced regulation of FAT1. Further studies are needed to elucidatewhetherIDH1-R132Hregulates FAT1 expression through transcriptional or post-translational mechanisms.
In addition tonormoxia, we also determined the effect of IDH1-R132H overexpression on proliferation, apoptosis and migration under hypoxia. However, we did not find significant differences between the effects of IDH1-R132H overexpression under normoxic and hypoxic conditions (data not shown). Kessler et al. also reported that IDH1-R132H mutation caused a less aggressive phenotype and radio-sensitized human glioblastoma cells independent of the oxygenation status. Collectively, it is suggested that IDH1-R132H-induced effect on GBM is not relied on oxygen status.
In summary, we identified that IDH1-R132H induced FAT1 expression, promoted the generation of ROS, increased HIF-1α expression, leading to the reduction of cell proliferation, increase of apoptosis and enhancement of chemosensitivity [Figure 7]. Our findings provide new insights into IDH1-R132H-regualted downstream signaling in GBM and highlight the importance of IDH1-R132H-FAT1-ROS-HIF-1α signaling pathway in potential therapeutic intervention of GBM.
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Conflicts of interest
There are no conflicts of interest.
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