Role of AMPK in UVB-induced DNA damage repair and growth control

Abstract

Skin cancer is the most common cancer in the United States, while DNA-damaging ultraviolet B (UVB) radiation from the sun remains the major environmental risk factor. Reducing skin cancer incidence is becoming an urgent issue. The energy-sensing enzyme 5'-AMP-activated protein kinase (AMPK) has a key role in the regulation of cellular lipid and protein metabolism in response to stimuli such as exercise and changes in fuel availability. However, the role of AMPK in the response of skin cells to UVB damage and in skin cancer prevention remains unknown. Here we show that AMPK activation is reduced in human and mouse squamous cell carcinoma as compared with normal skin, and by UVB irradiation, suggesting that AMPK is a tumor suppressor. At the molecular level, AMPK deletion reduced the expression of the DNA repair protein xeroderma pigmentosum C (XPC) and UVB-induced DNA repair. AMPK activation by its activators AICAR (5-aminoimidazole-4-carboxamide ribonucleoside) and metformin (N',N'-dimethylbiguanide), the most widely used antidiabetic drug, increased the expression of XPC and UVB-induced DNA repair in mouse skin, normal human epidermal keratinocytes, and AMPK wild-type (WT) cells but not in AMPK-deficient cells, indicating an AMPK-dependent mechanism. Topical treatment with AICAR and metformin not only delayed onset of UVB-induced skin tumorigenesis but also reduced tumor multiplicity. Furthermore, AMPK deletion increased extracellular signal-regulated kinase (ERK) activation and cell proliferation, whereas AICAR and metformin inhibited ERK activation and cell proliferation in keratinocytes, mouse skin, AMPK WT and AMPK-deficient cells, suggesting an AMPK-independent mechanism. Finally, in UVB-damaged tumor-bearing mice, both topical and systemic metformin prevented the formation of new tumors and suppressed growth of established tumors. Our findings not only suggest that AMPK is a tumor suppressor in the skin by promoting DNA repair and controlling cell proliferation, but also demonstrate previously unknown mechanisms by which the AMPK activators prevent UVB-induced skin tumorigenesis.

Fig. 1

AMPK pathway is inhibited in skin tumors from human and mouse and by UVB. A, immunoblot analysis of p-AMPK (T172) and β-actin in normal human skin and human SCC. B, immunoblot analysis of p-ACC (S79), ACC and GAPDH in sham- or UVB-irradiated non-tumor skin and UVB-induced skin tumors from SKH-1 mice. Mice were irradiated with UVB (100 mJ/cm²) three times a week for 23 weeks. Non-tumor skin or tumor was collected at 24 h after the final UVB irradiation or sham irradiation. C, immunoblot analysis of p-AMPK, AMPK, p-ACC, ACC and GAPDH in SKH-1 mouse skin sham-treated or treated with UVB at 0.5, 6 or 24 h post-UVB (100 mJ/cm²).

Fig. 2

AICAR and metformin enhance UVB-induced DNA repair through activating AMPK. A, slot blot analysis of the levels of CPD and 6-4PP in MEF cells (n=3) with wild-type AMPK (WT) or AMPK knockout (KO) at 0, 6, 24, and 48 h post-UVB (5 mJ/cm²). B, quantification of percentage (%) of CPD repair from A. *, P < 0.05, significant differences between AMPK WT and KO groups. C, slot blot analysis of the levels of CPD in SKH-1 mouse skin (n =3) treated with vehicle (Veh), AICAR, or metformin (Met) at different times post-UVB (100 mJ/cm²). D, quantification of percentage (%) of CPD repair from C. *, P < 0.05, significant differences between vehicle- and AICAR- or metformin-treated groups. E, slot blot analysis of the levels of 6-4PP in SKH-1 mouse skin (n =3) treated with vehicle (Veh), AICAR, or metformin (Met) at different times post-UVB (100 mJ/cm²). F, quantification of percentage (%) of 6-4PP repair from E. G, slot blot analysis of the levels of CPD and 6-4PP in WT or KO MEF cells (n=3) treated with vehicle (Veh), AICAR (AI, 1 mM), or metformin (2 mM) at 0, 6, 24, and 48 h post-UVB (5 mJ/cm²). H, immunoblot analysis of DDB1, DDB2, XPC, AMPK and GAPDH in WT and KO AMPK MEF cells. I, immunoblot analysis of XPC and GAPDH in NHEK cells treated with vehicle (Veh), AICAR (AI, 1 mM) or metformin (2 mM). Error bars in panels B, D and F indicate S.E.

Fig. 3

AICAR and metformin prevent UVB-induced skin tumorigenesis in SKH-1 hairless mice. A, immunoblot analysis of p-ACC, ACC, and GAPDH in SKH-1 mouse skin at 24 h after the final topical treatment with vehicle (Veh), AICAR (1 μmol), or metformin (Met, 2 μmol) for 23 weeks. B, percent (%) of tumor-free mice in vehicle (Veh), AICAR, or metformin-treated mice following sham or UVB irradiation (n=10). SKH-1 mice were treated with topical AICAR (1 μmol) or metformin (2 μmol) 1 h prior to each UVB irradiation (100 mJ/cm²) three times a week for 23 weeks. C, Average number (#) of tumors per mouse from mice treated as in B. D, average number (#) of large (> 1cm in diameter) and small (< 1cm in diameter) tumors per mouse. *, P < 0.05, significant differences between vehicle- and AICAR- or metformin-treated groups. Error bars in panel D indicate S.E.

Fig. 4

AICAR and metformin reduce cell proliferation in mouse skin and MEF cells independent of the AMPK pathway. A, histological analysis of non-tumor mouse epidermis (n = 10) topically treated with vehicle, AICAR (1 μmol) or metformin (2 μmol) for 23 weeks post-UVB or –sham. Scale Bar: 200 μm. B, quantification of epidermal thickness (μm) in A. C, immunohistochemical analysis of Ki67-positive cells in mouse skin (n = 5) topically treated with vehicle, AICAR (1 μmol) or metformin (2 μmol) for 23 weeks post-UVB or –sham irradiation. Scale Bar: 50 μm. D, quantification of Ki67-positive (Ki67+) cells in C. *, P < 0.05, significant differences between vehicle- and AICAR/metformin-treated groups. E, proliferation analysis using the MTS assay (Promega) in WT or KO AMPK MEF cells. *, P < 0.05, significant differences between AMPK WT and KO cells. F, proliferation analysis using the MTS assay (Promega) in WT or KO AMPK MEF cells treated with vehicle, AICAR (AI, 1 mM) or metformin (Met, 2 mM). *, P < 0.05, significant differences between vehicle- and AI/Met-treated groups in WT and KO cells. Error bars in panels B, D, E, and F indicate S.E.

Fig. 5

AMPK is not required for inhibiting the ERK pathway by AICAR and metformin. A, immunoblot analysis of p-ACC, ACC, p-ERK, ERK, and GAPDH in mouse skin at 24 h after the final topical treatment with vehicle, AICAR (1 μmol) or metformin (2 μmol) for 23 weeks. B, immunoblot analysis of Cyclin D1, p-ERK, ERK, p-ACC and GAPDH in NHEK cells at 24 h after treatment with vehicle, AICAR (1 mM) or metformin (2 mM). C, immunoblot analysis of AMPK, p-ERK, ERK, p-EGFR, cyclin D1 and GAPDH in AMPK WT and KO MEF cells. D, immunoblot analysis of cyclin D1, p-ERK, ERK, p-EGFR, AMPK, and GAPDH in KO MEF cells treated with vehicle (−), PD (PD98059, 20 μM) and AG (AG1478, 1 μM), and WT MEF cells. E, immunoblot analysis of p-ERK, ERK, p-EGFR, AMPK and GAPDH in WT and KO MEF cells treated with vehicle, AICAR (1 mM), or metformin (2 mM).

Fig. 6

Metformin prevents new tumor formation and suppresses growth of established tumors in mice. A, a schematic diagram of the experimental design for B-F, in which mice were treated with topical metformin (Met-T, 2 μmol) or systemic metformin (Met-G, 300 mg/kg body weight) 1 h prior to each UVB treatment at 17 weeks after the initial UVB irradiation, together with continuing UVB irradiation three times a week for 8 weeks. B, representative mouse pictures from experimental design as in A. C, immunoblot analysis of p-ACC, ACC and GAPDH. D, number (#) of new tumors per mouse at different weeks following metformin treatment as in A (n = 3). E, average volume (mm³) of established tumors formed at 17 weeks post-UVB at different weeks following treatment as in A. F. histological analysis of non-tumor (NT) epidermis treated with metformin as in A for 8 weeks by hematoxylin and eosin stain (H&E) and immunohistochemical analysis of Ki67-positive (Ki67+) cells in non-tumor (NT) and skin tumors. *, P < 0.05, significant differences between vehicle- and metformin-treated groups. Error bars in panels D and E indicate S.E.