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, 12 (2), 357-68

Autophagy Positively Regulates DNA Damage Recognition by Nucleotide Excision Repair

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Autophagy Positively Regulates DNA Damage Recognition by Nucleotide Excision Repair

Lei Qiang et al. Autophagy.

Abstract

Macroautophagy (hereafter autophagy) is a cellular catabolic process that is essential for maintaining tissue homeostasis and regulating various normal and pathologic processes in human diseases including cancer. One cancer-driving process is accumulation of genetic mutations due to impaired DNA damage repair, including nucleotide excision repair. Here we show that autophagy positively regulates nucleotide excision repair through enhancing DNA damage recognition by the DNA damage sensor proteins XPC and DDB2 via 2 pathways. First, autophagy deficiency downregulates the transcription of XPC through TWIST1-dependent activation of the transcription repressor complex E2F4-RBL2. Second, autophagy deficiency impairs the recruitment of DDB2 to ultraviolet radiation (UV)-induced DNA damage sites through TWIST1-mediated inhibition of EP300. In mice, the pharmacological autophagy inhibitor Spautin-1 promotes UVB-induced tumorigenesis, whereas the autophagy inducer rapamycin reduces UVB-induced tumorigenesis. These findings demonstrate the crucial role of autophagy in maintaining proper nucleotide excision repair in mammalian cells and suggest a previously unrecognized tumor-suppressive mechanism of autophagy in cancer.

Keywords: DDB2; UV; XPC; autophagy; nucleotide excision repair.

Figures

Figure 1.
Figure 1.
Autophagy deficiency inhibits nucleotide excision repair and downregulates XPC. (A, B) Slot blot analysis of the levels of CPD (A) and 6-4PP (B) in WT and atg5 KO MEF cells at 0, 12, 24 and 48 h post-UVB (10 mJ/cm2) for CPD and 0, 1 and 3 h post-UVB (10 mJ/cm2) for 6-4PP. (C, D) Quantification of percentage (%) of CPD repair (C) from A and 6-4PP repair (D) from B (mean±SD, n=3). *, P < 0.05, compared with WT group, Student t test (C, D). (E) Immunoblot analysis of XPC and GAPDH in WT and atg5 KO MEF and iBMK cells. (F) Immunoblot analysis of XPC, LC3-I/II, ATG5, ATG7 and GAPDH in iBMK cells lentivirally infected with shRNA nontargeted control (shCon) or shRNA targeting Atg5 (shAtg5) or Atg7 (shAtg7). (G) Immunoblot analysis of XPC, UB-XPC (polyubiquitinated XPC), LC3-I, LC3-II, ATG7 and GAPDH in HaCaT cells transfected with shCon or shATG7 at 0, 0.5, 1.5, 6 and 24 h post-UVB (20 mJ/cm2). The results were obtained from 3 independent experiments.
Figure 2.
Figure 2.
Autophagy deficiency links TWIST1 to suppression of nucleotide excision repair. (A) Immunoblot analysis of XPC, TWIST1, SQSTM1 and GAPDH in WT and atg5 KO MEF cells lentivirally infected with shCon or 2 independent shRNAs targeting Twist1 (shTwist1 #1 or shTwist1 #2). (B) Immunoblot analysis of XPC, TWIST1, SQSTM1 and GAPDH in WT and sqstm1 KO MEF cells lentivirally infected with vector control (Con) or Myc-Twist1 expression vector. (C, D) Slot blot analysis of the levels of CPD (C) and 6-4PP (D) in WT and atg5 KO MEF cells lentivirally infected with shCon or shTwist1. Cells were collected for analysis at 0, 24 and 48 h post-UVB (10 mJ/cm2) for CPD and 0, 1 and 3 h post-UVB (10 mJ/cm2) for 6-4PP. (E, F) Quantification of percentage (%) of CPD repair (E) from C and 6-4PP repair (F) from D (mean±SD, n=3). *, P < 0.05, compared with WT group; #, P < 0.05, compared with KO+shCon group, Student t test. The results were obtained from 3 independent experiments.
Figure 3.
Figure 3.
Autophagy deficiency inhibits XPC transcription through the TWIST1-AKT pathway. (A) RT-PCR analysis of Xpc mRNA levels in WT and atg5 KO MEF cells lentivirally infected with shCon or shTwist1 (mean±SD, n=3). (B) Luciferase reporter assay of the Xpc promoter in WT and atg5 KO MEF cells lentivirally infected with shCon or shTwist1 (mean±SD, n=3). (C) Luciferase reporter assay of the Xpc promoter with wild-type (WT) sequence, deletion of E-Box2/3, mutation of E-Box1, mutation of E-Box4 or deletion of E-Box2/3 in combination with mutation of E-Box1/3 in WT and atg5 KO MEF cells (mean±SD, n=3). *, P < 0.05, compared with WT group; #, P < 0.05, compared with WT or shCon group, Student t test (A-C). (D) Immunoblot analysis of p-AKT (Ser473), total AKT, TWIST1 and GAPDH in WT and atg5 KO MEF cells lentivirally infected with shCon or shTwist1. (E) Immunoblot analysis of RBL2, E2F4, LMNB/LAMIN B and GAPDH in cytosolic and nuclear fraction from WT and atg5 KO MEF cells. (F) Luciferase reporter assay of the Xpc promoter with wild-type (WT) sequence and E2F mutation (E2F mut) in WT and atg5 KO MEF (mean±SD, n=3). (G) Immunoblot analysis of XPC, p-AKT (Ser473), total AKT, TWIST1 and GAPDH in WT and atg5 KO MEF cells treated with or without the PI3K-AKT pathway inhibitor LY294002 (LY, 10 μM). (H, I) Quantification of percentage (%) of CPD repair (H) and 6-4PP repair (I) in WT and atg5 KO MEF cells treated with or without LY294002 (LY, 10 μM) (mean±SD, n=3). *, P < 0.05, compared with WT group; #, P < 0.05, compared with Veh group, Student t test (H, I). The results were obtained from 3 independent experiments.
Figure 4.
Figure 4.
Autophagy deficiency inhibits 6-4PP repair via decreasing XPC while it inhibits CPD repair via both decreasing XPC availability and damage recognition by DDB2. (A) Immunoblot analysis of XPC, TWIST1 and GAPDH in WT and atg5 KO MEF cells transfected with a construct expressing vector control (Con) or XPC. (B, C) Quantification of percentage (%) of CPD repair (B) and 6-4PP repair (C) in WT and atg5 KO MEF cells transfected with Con or XPC (mean±SD, n=3). *, P < 0.05, compared with WT group; #, P < 0.05, compared with KO+Con group, Student t test. (D) Immunoblot analysis of XPC, TWIST1 and GAPDH in XPC reconstituted cells (XPC-/--CMV-XPC) transfected with Con and MYC-TWIST1. (E) Quantification of percentage (%) of CPD repair in XPC-/--CMV-XPC cells transfected with Con and MYC-TWIST1 (mean±SD, n=3). *, P < 0.05, compared with Con group. (F, H) Immunofluorescence assay of the colocalization of XPC (F) and DDB2 (H) with subnuclear CPD in HaCaT cells transfected with Con or MYC-TWIST1 at 0.5 h post-UV (10 mJ/cm2) through a 5 μm micropore filter. Scale bar: 10 μm. (G, I) The relative intensity of XPC (G) and DDB2 (I) foci was calculated by analyzing 100 foci and normalized to that of CPD (n =100, error bar: SD). The results were obtained from 3 independent experiments.
Figure 5.
Figure 5.
Autophagy deficiency inhibits DDB2 recruitment through Twist1 binding and inhibition of EP300. (A) Immunoprecipitation was performed in WT and atg5 KO MEF cells with the indicated antibodies, followed by immunoblot analysis of EP300 and TWIST1. (B) Immunoblot analysis of EP300, XPC, TWIST1 and GAPDH in WT and atg5 KO MEF cells transfected with Con or the combination of Xpc and Ep300. (C) Quantification of percentage (%) of CPD repair in WT and atg5 KO MEF cells transfected with Con or the combination of Xpc and Ep300 (mean±SD, n=3). *, P < 0.05, compared with WT group; #, P < 0.05, compared with Con group, Student t test. (D) Immunoblot analysis of XPC, TWIST1 and GAPDH in XPC-/--CMV-XPC cells transfected with Con and MYC-TWIST1 (36 to 72) deletion. (E) Slot blot analysis of the levels of CPD in XPC-/--CMV-XPC cells transfected with Con and MYC-TWIST1 (36 to 72) deletion. (F) Quantification of percentage (%) of CPD repair from (E) (mean±SD, n=3). *, P < 0.05, compared with WT group; #, P < 0.05, compared with Con group, Student t test. (G) Immunofluorescence assay of the colocalization of DDB2 with subnuclear CPD in HaCaT cells transfected with Con or MYC-TWIST1 (36 to 72) deletion at 0.5 h post-UV (10 mJ/cm2) through a 5-μm micropore filter. Scale bar: 10 μm. (H) The relative intensity of DDB2 was calculated by analyzing 100 foci and normalized to that of CPD (n =100, Mean±SD). All results were obtained from 3 independent experiments.
Figure 6.
Figure 6.
Pharmacological modulators of autophagy regulate UVB-induced skin carcinogenesis. (A) A schematic diagram of the experimental design for B to G, in which mice were treated with UVB irradiation for 17 wk, 3 times per wk, and then with vehicle only or topical rapamycin (Rap, 10 nmol) or Spautin-1 (SP, 25 nmol) 3 h prior to each UVB or sham treatment 3 times a wk for another 10 wk. (B) Immunoblot analysis of LC3-I, LC3-II, SQSTM1, XPC, TWIST1 and GAPDH in mouse skin collected 12 h after the final treatment. (C) Representative histological and immunohistochemical analysis of SQSTM1, TWIST1, and MKI67 protein levels (brown) in UVB-irradiated mouse skin treated with Vehicle (Veh), rapamycin, or Spautin-1. Scale bar: 50 µm. (D, E) Number (#) of new tumors per mouse at different weeks following Rap or Spautin-1 treatment as in (A) (n=5), without (D) or with (E) continuing UVB irradiation. (F, G) Average volume (mm3) of established tumors formed at 17 wk post-UVB at different weeks following treatment as in (A), without (F) or with (G) continuing UVB irradiation. The results were mean±SD (n=5). *, P <0.05; **, P<0.01 compared with the Veh group in D to G). [Change label to Spautin-1.]
Figure 7.
Figure 7.
Schematic diagram of autophagy regulation of nucleotide excision repair in skin carcinogenesis. Autophagy positively regulates nucleotide excision repair through decreasing TWIST1 stability, by which autophagy (1) maintains the proper transcription of XPC through inhibiting the transcription repressor complex E2F4-RBL2 and (2) positively regulates the recruitment of DDB2 to UV-induced DNA damage sites through EP300. Thus autophagy may reduce UV-induced accumulation of mutations, thereby reducing skin tumorigenesis.

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