Fstl1/DIP2A/MGMT signaling pathway plays important roles in temozolomide resistance in glioblastoma

Temozolomide was recognized as the first-line therapy for glioblastoma to prolong the survival of patients noticeably, while recent clinical studies found that some patients were not sensitive to temozolomide treatment. The possible mechanisms seemed to be methylguanine-DNA-methyltransferase (MGMT), mismatch repair, PARP, etc. And the abnormal expression of MGMT might be the most direct factor. In this study, we provide evidence that Fstl1 plays a vital role in temozolomide resistance by sequentially regulating DIP2A protein distribution, H3K9 acetylation (H3K9Ac), and MGMT transcription. As a multifunctional protein widely distributed in cells, DIP2A cooperates with the HDAC2–DMAP1 complex to enhance H3K9Ac deacetylation, prevent MGMT transcription, and increase temozolomide sensitivity. Fstl1, a glycoprotein highly expressed in glioblastoma, competitively binds DIP2A to block DIP2A nuclear translocation, so as to hinder DIP2A from binding the HDAC2–DMAP1 complex. The overexpression of Fstl1 promoted the expression of MGMT in association with increased promoter H3K9Ac. Upregulation of Fstl1 enhanced temozolomide resistance, whereas Fstl1 silencing obviously sensitized GBM cells to temozolomide both in vivo and in vitro. Moreover, DIP2A depletion abolished the effects of Fstl1 on MGMT expression and temozolomide resistance. These findings highlight an important role of Fstl1 in the regulation of temozolomide resistance by modulation of DIP2A/MGMT signaling.


manufacturer's instructions. For stable transfection, GBM cells were transfected with
Fstl1, shDIP2A or shFstl1 (Genechem, Shanghai, China) lentiviral particles according to the manufacturer's protocol. Scrambled lentiviral particles were used as a control.
After 48h 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 Fstl1 or DIP2A expression.

Methylation-specific PCR assay
The methylation status of the MGMT promoter was evaluated by methylation-specific PCR (MSP) assay as described previously 4,5 . Genomic DNA was extracted from the cells using a QIAamp DNA Mini Kit (Qiagen). Sodium bisulfite conversion of 200 ng of the purified DNA was performed using an EpiTect Bisulfite Kit (Qiagen) according to the manufacturer's protocol. MSP of bisulfate converted DNA was carried out in a nested, two-stage PCR approach using GeneAmp PCR System 2700 (Applied Biosystems, Grand Island, NY). Amplified PCR products were separated by 3% agarose gel electrophoresis and visualized with ethidium bromide.

Immunofluorescence staining
Cells grown on confocal dishes (Cellvis, CA, USA) were fixed for 30 min in 4% paraformaldehyde at room temperature, and then incubated in PBS containing 0.1%

Co-immunoprecipitation (Co-IP)
Cells were lysed in SDS lysis buffer on ice for 30 min, and the supernatants were collected by centrifugation at 4°C and 14,000 × g for 15min. The cleared protein lysates were then incubated with primary antibodies with rotation overnight at 4°C.
Then, the mixtures were incubated with immobilized protein A/G beads (Thermo Scientific) with rotation at 4°C for 2h. The beads were collected by centrifugation at 3,000×g for 2min, and then washed five times with 0.5ml IP wash buffer. SDS loading buffer was added to the beads, and the samples were denatured at 95°C for 8-10min. Finally, the supernatants were collected and stored at −80°C or immediately analyzed by WB. 7

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

Western blotting
Western blotting (WB) was performed as described previously 6 , and images were captured by Bio-Rad ChemiDoc XRS+ (Bio-Rad, CA, USA). The primary antibodies used are listed in the supplemental experimental procedures.