BACH1 Promotes Temozolomide Resistance in Glioblastoma through Antagonizing the Function of p53

The acquisition of drug resistance is a persistent clinical problem limiting the successful treatment of glioblastoma (GBM). However, the molecular mechanisms by which initially chemoresponsive tumors develop therapeutic resistance remain poorly understood. In this study, we report that BACH1, a heme-binding protein that participates in transcriptional repression or activation, was significantly upregulated in glioblastoma tissues. Overexpression of BACH1 in GBM cells conferred resistance to temozolomide, whereas its inhibition markedly sensitized resistant cells to temozolomide in vitro and in vivo. Further investigation revealed that BACH1 activation significantly enhanced the expression of MGMT, and depletion of p53 disrupted the effects of BACH1 on MGMT and temozolomide resistance. P53 sequesters SP1 to prevent its binding to the MGMT promoter region and thus inhibits MGMT expression. Moreover, BACH1 overexpression impaired the association between p53 and SP1 via competitive binding p53, and antagonized the impact of p53 on MGMT expression. Finally, we found that BACH1 low expression correlated with better prognosis in GBM patients undergoing temozolomide therapy, especially in patients with wild-type TP53. Collectively, our findings identify a potential mechanism by which wild-type TP53 GBM cells develop resistance to temozolomide and suggest that targeting this pathway may be beneficial for overcoming resistance.


Patients and samples
A total of 26 patients (17 males and 9 females) with a median age of 49.6 years (range, 21-67years) who had been treated surgically at the Department of Neurosurgery, The First Affiliated Hospital of Nanjing Medical University (Nanjing, China), between January 2012 and October 2013 were selected for the study. Among the 26 cases, 21 tumor samples and five non-tumorous brain specimens were obtained for analysis from the patients registered at the hospital. Informed consent was obtained from each subject, and the study was approved by the ethics committee of the hospital.
The glioma tissue samples were histologically diagnosed by the Department of Pathology at The First Affiliated Hospital of Nanjing Medical University according to the WHO classification 1 . As controls, five human non-tumor brain tissue (NBT) samples were obtained primarily from the cortex of patients with decompressive surgery after physical injury to the brain. Samples were immediately snap-frozen in liquid nitrogen until use. All methods were performed in accordance with the approved guidelines.

Cell culture and reagents
The human glioblastoma multiforme (GBM) cell lines A172, U87, U251, LN229, U138 and T98 were purchased from Shanghai Cell Bank of the Chinese Academy of Sciences (Shanghai, China). DBTRG-05MG cell line was bought from Sigma-Aldrich

Plasmids construction, transfection and stable cell establishment
The entire coding sequence of BACH1 and p53 was obtained from HUVEC mRNA by RT-PCR. BACH1 and p53 cDNA were purified by Genechem (Shanghai, China) and were cloned into pcDNA3.1-FLAG vector to generate pcDNA3.1-FLAG-BACH1 and pcDNA3.1-FLAG-p53 recombinant plasmid. Recombinant plasmids expressing FLAG-p53-mutants were provided by Genechem (Shanghai, China). Transient transfection was performed using Lipofectamine™ 2000 transfection reagent (Invitrogen) according to the manufacturer's instructions. For stable transfection, GBM cells were transfected with BACH1, shBACH1, or shp53 (Genechem) lentiviral particles according to the manufacturer's protocol. Scrambled lentiviral particles were used as a control. After 48 h of incubation, the medium was replaced with DMEM containing 5μg/ml puromycin. After maintenance for 3-4 weeks in selection media, puromycin-resistant colonies were selected and screened for BACH1 or p53 expression.

Chromatin immunoprecipitation
Chromatin immunoprecipitation (ChIP) assays were performed using the EZ-magna ChIP kit (Millipore, Bedford, MA, USA) according to the manufacturer's protocol.
Control samples were immunoprecipitated with 5 μg IgG (Millipore). After immunoprecipitation, the beads were washed sequentially with low-salt buffer, high-salt buffer, LiCl buffer, and TE buffer each for 5 min at 4˚C. The immunoprecipitated DNA was then eluted by incubation in 100 μl of elution buffer (0.1M NaHCO3 and 1% SDS) containing 10 μg proteinase K (Millipore) at 62°C for 2 h with rotation. The eluted DNA was purified using the columns and buffers contained in the kit (Millipore), and was finally re-dissolved in 50 μl of PCR-grade water. The eluted DNA was subjected to quantitative PCR using SYBR Green master mix (Roche Applied Science, Upper Bavaria, Germany).