Regulation of FANCD2 by the mTOR pathway contributes to the resistance of cancer cells to DNA double-strand breaks

Abstract

Deregulation of the mTOR pathway is closely associated with tumorigenesis. Accordingly, mTOR inhibitors such as rapamycin and mTOR-selective kinase inhibitors have been tested as cancer therapeutic agents. Inhibition of mTOR results in sensitization to DNA-damaging agents; however, the molecular mechanism is not well understood. We found that an mTOR-selective kinase inhibitor, AZD8055, significantly enhanced sensitivity of a pediatric rhabdomyosarcoma xenograft to radiotherapy and sensitized rhabdomyosarcoma cells to the DNA interstrand cross-linker (ICL) melphalan. Sensitization correlated with drug-induced downregulation of a key component of the Fanconi anemia pathway, FANCD2 through mTOR regulation of FANCD2 gene transcripts via mTORC1-S6K1. Importantly, we show that FANCD2 is required for the proper activation of ATM-Chk2 checkpoint in response to ICL and that mTOR signaling promotes ICL-induced ATM-Chk2 checkpoint activation by sustaining FANCD2. In FANCD2-deficient lymphoblasts, FANCD2 is essential to suppress endogenous and induced DNA damage, and FANCD2-deficient cells showed impaired ATM-Chk2 and ATR-Chk1 activation, which was rescued by reintroduction of wild-type FANCD2. Pharmacologic inhibition of PI3K-mTOR-AKT pathway in Rh30 rhabdomyosarcoma cells attenuated ICL-induced activation of ATM, accompanied with the decrease of FANCD2. These data suggest that the mTOR pathway may promote the repair of DNA double-strand breaks by sustaining FANCD2 and provide a novel mechanism of how the Fanconi anemia pathway modulates DNA damage response and repair.

Figure 1. The mTOR Pathway Promotes Cell Survival in Response to Mephalan by Sustaining FANCD2

A, 500 cells were plated in 10 cm dish for 6 hr, then AZD8055 (2 μM) was applied for 16 hr. Melphalan (0.2 μg/mL) was added to untreated or AZD8055 treated cells for 2 hr. The drugs were washed away. Two weeks later, the colonies were counted. Shown was a representative of three independent experiments. B, quantification of the colonies in Figure 1A. Error bars: Mean ± SD (n=3). C, Rh30 cells were treated with AZD8055 (2 μM) for 16 hr. Total proteins were extracted for immunoblotting. D, lymphoblast PD20 cells derived from a Fanconi anemia D2 patient were stably transfected with empty vector (pMMP-Puro) or wild type FANCD2 plasmid (pMMP-wt-FANCD2). FANCD2 and γH2AX were detected by immunoblotting. (−), pMMP-Puro; (+), pMMP-wt-FANCD2. E, Rh30 cells were transfected with control or FANCD2 siRNA. 48 hr later, total proteins were extracted for immunoblotting. F, Rh30 cells were transfected with vector or FANCD2 plasmid. 24 hr later, AZD8055 (2 μM) was added for additional 24 hr. Total proteins were extracted for immunoblotting. G, Rh30 cells were transfected with control or mTOR siRNA. 48 hr later, total proteins were extracted for immunoblotting. H, Rh30 cells were treated with AZD8055 (AZD, 2 μM) or melphalan (MP, 2 μg/ml) for the time indicated. Total proteins were extracted for immunoblotting. UT, untreated. Upper band of FANCD2 staining showed the monoubiquitination of FANCD2. GAPDH and β-Actin served as loading controls.

Figure 2. FANCD2 is Controlled by mTOR Signaling in Pediatric Rhabdomyosarcoma in vivo

A and B, pediatric rhabdomyosarcoma Rh10 and Rh30 tumor xenograft models were propagated subcutaneously in SCID mice and were treated with mTOR kinase inhibitor AZD8055 at 20 mg/kg/day. Tumors were harvested 1, 4, 8 and 24hr post treatment on day 4 and were pulverized under liquid N_2. Total proteins were extracted for immunoblotting. C, mice bearing subcutaneous rhabdomyosarcoma xenografts, Rh18, Rh10 and Rh30 tumor xenografts were treated with mTOR kinase inhibitor AZD8055 (20 mg/kg daily). Tumors were harvested 24 hr after the fourth dose administered. Total proteins were extracted for immunoblotting to detect FANCD2. D, pediatric rhabdomyosarcoma Rh18 and Rh30 tumor xenograft models were propagated subcutaneously in SCID mice and were treated with rapamycin at 5 mg/kg per day. Tumors were harvested 24 hr post treatment on day 1. Total proteins were extracted for immunoblotting.

Figure 3. The mTOR Pathway Regulates FANCD2 at Transcription Level

A, Rh30 cells were treated with AZD8055 (2 μM) for the times indicated. The total RNA was extracted to detect FANCD2 mRNA by real-time RT-PCR with GAPDH as internal control. Relative quantity (Ln) of FANCD2 mRNA was plotted. B, Rh30 cells were treated with rapamycin or MK2206 for 12 hr. Total RNA was extracted to detect FANCD2 mRNA by real-time RT-PCR with GAPDH as internal control. Relative quantity (Ln) of FANCD2 mRNA was plotted. C, Rh30 cells were transfected with vector or AKT1-wt plasmid. 48 hr later, the total RNA was extracted to detect FANCD2 mRNA by real-time RT-PCR with GAPDH as internal control. Relative quantity (Ln) of FANCD2 mRNA was plotted. D, Rh30 cells were treated with AZD8055 for 16 hr. Total proteins were extracted for immunoblotting. E, Rh30 cells were treated with rapamycin, AZD8055, MK2206 or PD03332991 (1 μM) for 16 hr. Total proteins were extracted for immunoblotting. F, Rh30 cells were treated with PD03332991 for 12 hr. The total RNA was extracted to detect FANCD2 mRNA by real-time RT-PCR with GAPDH as internal control. Relative quantity (Ln) of FANCD2 mRNA was plotted. G, wild type (MEF WT) and S6K1 KO MEF cells were treated with rapamycin (100 ng/mL) for 24 hr. Total proteins were extracted to detect FANCD2 by immunoblotting with beta actin as loading controls. H, cells were treated as in G, total mRNA was extracted to detect mouse FANCD2 mRNA by real-time RT-PCR with GAPDH as internal control. Relative quantity (Ln) of FANCD2 mRNA was plotted. Error bars: Mean ± SD (n=3).

Figure 4. FANCD2 is Required for ATR-Chk1 and ATM-Chk2 Activation in Response to DNA Replications Stress and Damage in Lymphoblast PD20 Cells

A, total protein extracts from lymphoblast PD20 cells from Fanconi anemia D2 patient stably transfected with empty vector (pMMP-Puro) or wild type FANCD2 plasmid (pMMP-wt-FANCD2) were used for immunoblotting. TU, untreated. B and C, lymphoblast PD20 cells as in A were treated with hydroxyurea (HU, 2 mM) or melphalan (MP, 2 μg/mL) for 6 hr, total protein extracts were for immunoblotting. −, pMMP-Puro; +, pMMP-wt-FANCD2.

Figure 5. ATR-Chk1 Precedes the Activation of ATM-Chk2 in Response to ICL

A, Rh30 cells were treated with 2 μg/mL melphalan for the time as indicated. Total proteins were extracted for immunoblotting to detect Chk1, pChk1-S345, γH2AX and PARP1. B, Rh30 cells were treated with 2 μg/mL melphalan for the time as indicated. Total proteins were extracted for immunoblotting to detect pChk1-S345, ATM and pATM-S1981. C, Rh30 cells were transfected with control or FANCD2 siRNA. 48 hr later, melphalan with the indicated concentrations was added for 8 hr. Total proteins were extracted for immunoblotting of γH2AX, pChk1-S345 and FANCD2. D, Rh30 cells were transfected with FANCD2 siRNA for 48 hr or AZD8055 (2 μM) for 16 hr. 2 μg/mL melphalan was added alone or in the cells with AZD8055 or FANCD2 siRNA for 8 hr. FANCD2 and γH2AX was detected by immunocytochemistry. DAPI stained nuclei. Scare bar: 20 μM.

Figure 6. The mTOR Pathway is Required for ATM-Chk2 Checkpoint Activation in Response to DNA Interstrand Crosslinker Induced DNA Damage

A, lymphoblast PD20 cells stably transfected with pMMP-Puro or pMMP-wt-FANCD2 were treated with AZD8055 (2 μM) for 16 hr, then melphalan (MP, 2 μg/mL) was added alone or in combination with AZD8055 and incubated for 6 hr. Immunoblotting was done to detect FANCD2, Chk2, and pChk2-T68. −, pMMP- +, pMMP-wt-FANCD2. B, Rh30 cells were treated with rapamycin (100 ng/mL), AZD8055 (2 μM) or MK2206 (10 μM) for 16 hr. Then melphalan (MP, 2 μg/mL) was added alone or in combination as indicated and incubated for 6 hr.Immunoblotting was done to detect FANCD2 and pATM-S1981. C, Rh30 cells were treated with rapamycin (100 ng/mL), AZD8055 (2 μM) or MK2206 (10 μM) for 16 hr. Then melphalan (MP, 2 μg/mL), cisplatin (5 μM), and mitomycin C (2 μM) was added alone or in combination as indicated for 6 hr. 2 hr prior to protein extraction, MG132 (2 μM) was added as indicated. FANCD2 and pChk2-T68 were detected by Immunoblotting. GAPDH served as loading control.