PTEN positively regulates UVB-induced DNA damage repair

Abstract

Nonmelanoma skin cancer is the most common cancer in the United States, where DNA-damaging ultraviolet B (UVB) radiation from the sun remains the major environmental risk factor. However, the critical genetic targets of UVB radiation are undefined. Here we show that attenuating PTEN in epidermal keratinocytes is a predisposing factor for UVB-induced skin carcinogenesis in mice. In skin papilloma and squamous cell carcinoma (SCC), levels of PTEN were reduced compared with skin lacking these lesions. Likewise, there was a reduction in PTEN levels in human premalignant actinic keratosis and malignant SCCs, supporting a key role for PTEN in human skin cancer formation and progression. PTEN downregulation impaired the capacity of global genomic nucleotide excision repair (GG-NER), a critical mechanism for removing UVB-induced mutagenic DNA lesions. In contrast to the response to ionizing radiation, PTEN downregulation prolonged UVB-induced growth arrest and increased the activation of the Chk1 DNA damage pathway in an AKT-independent manner, likely due to reduced DNA repair. PTEN loss also suppressed expression of the key GG-NER protein xeroderma pigmentosum C (XPC) through the AKT/p38 signaling axis. Reconstitution of XPC levels in PTEN-inhibited cells restored GG-NER capacity. Taken together, our findings define PTEN as an essential genomic gatekeeper in the skin through its ability to positively regulate XPC-dependent GG-NER following DNA damage.

Fig. 1

PTEN hemizygosity is a predisposing factor for skin tumorigenesis following low-level UVB radiation. A, percent (%) of tumor-free mice (n = 15). B, immunoblot analysis of PTEN and β-actin in normal +/+ and +/- skin, papillomas (PAP), and SCCs. C, immunohistochemical analysis of PTEN in sham-irradiated normal skin and UVB-induced tumor samples from Pten+/- mice using an anti-PTEN antibody. Scale bar: 20 μm.

Fig. 2

PTEN expression is significantly reduced in human skin tumors. A and B, representative immunohistochemical analysis of PTEN protein levels (brown) in normal skin and SCC. EP, epidermis; HF, hair follicle; T, tumor. Scale bar: 100 μm. C, Percent of tumors (in stacked column format) for each score of PTEN expression.

Fig. 3

PTEN is required for efficient GG-NER. A and B, immunoblot analysis of PTEN and β-actin (equal loading control) in NC and shPTEN cells (A) and in NC and siPTEN cells (B). C-E, Enzyme-linked immunosorbent assay (ELISA) of percent (%) repair of CPDs (C and D) and 6-4PPs (E) at intervals post-UVB (20 mJ/cm²). C, HaCaT cells infected with a retroviral vector expressing negative control (NC) or shRNA targeting PTEN (shPTEN). D and E, HaCaT cells transfected with negative control (NC) or siRNA targeting PTEN (siPTEN). Error bars show standard error (SE). *, P < 0.05, significant differences between shPTEN and NC cells, or PTEN and NC cells.

Fig. 4

PTEN inhibition delays exit from growth arrest post-UVB. A, quantification of percent (%) of BrdU positive (BrdU+) epidermal keratinocytes in Pten+/+ or Pten+/- mouse epidermis at different times post-sham-or UVB-irradiation. Error bars show standard error (SE). *, P < 0.05, significant difference between Pten+/+ and Pten+/- mouse epidermis. B, immunoblot analysis of PTEN, p-Chk1 (serine 345), Chk1, p-Chk2 (threonine 68), Chk2, γ-H2AX, and β-actin at 0.5, 1.5 and 6 h post-UVB (5 or 20 mJ/cm²) in HaCaT cells stably infected with a retroviral vector expressing negative control (NC) or shRNA targeting PTEN (shPTEN). C, immunoblot analysis of PTEN, p-AKT, AKT, p-Chk1, Chk1, p-Chk2, Chk2, γ-H2AX, p53, and GAPDH in NHEK cells transfected with negative control (NC) or siPTEN. D, immunoblot analysis of p-AKT, AKT, p-Chk1, Chk1, p-Chk2, Chk2, γ-H2AX, p53, and GAPDH in NHEK cells infected with an adenoviral vector expressing empty vector (EV) or constitutively active AKT (A+). E, immunoblot analysis of p-AKT, AKT, p-Chk1, Chk1, p-Chk2, Chk2, γ-H2AX, p53, and GAPDH in vehicle or LY294002 (LY, 10 μM)-treated NHEK cells transfected with siPTEN.

Fig. 5

PTEN loss suppresses XPC expression through increasing AKT signaling. A, immunoblot analysis of XPC, DDB2, DDB1, PTEN, and β-actin (equal loading control) in NC and shPTEN cells. B, immunoblot analysis of XPC, PTEN, and β-actin in HaCaT cells transfected with siRNA targeting PTEN (siPTEN) or negative control (NC). C, immunoblot analysis of XPC, PTEN and GAPDH in +/+ and +/- mouse epidermis. D. immunoblot analysis of XPC, DDB1, DDB2, and Histone H3 (loading control) in soluble and/or chromatin-bound fractions from NC and shPTEN cells at different times (5, 10, and 30 min) post-UVB (20 mJ/cm²). E, quantification of XPC levels in D. Error bars show standard error (SE). * in E, P < 0.05, significant difference between NC and shPTEN cells. F, luciferase reporter assay of XPC promoter in NC and shPTEN cells. G, immunoblot analysis of XPC, PTEN, and β-actin in shPTEN cells infected with an adenoviral vector expressing wild-type PTEN at different multiplicity of infection (MOI). H, Immunoblot analysis of XPC, p-AKT, AKT, and β-actin in shPTEN cells treated with vehicle or LY294002 (LY, 10 μM), as well as in NC HaCaT cells. I, Immunoblot analysis of XPC, PTEN, p-AKT, AKT, and GAPDH in siPTEN-transfected cells treated with vehicle or LY294002 (LY, 10 μM), as well as in NC-transfected NHEK cells. J, immunoblot analysis of XPC, PTEN, and β-actin in HaCaT cells transfected with or without the siRNA targeting PTEN and the XPC plasmid. K, enzyme-linked immunosorbent assay (ELISA) of percent (%) repair of CPDs from cells in J at different times post-UVB (20 mJ/cm²).

Fig. 6

Inhibition of p38 signaling by AKT is critical for inhibiting GG-NER by PTEN down-regulation. A, immunoblot analysis of p-p38, p38, PTEN, p-AKT (serine 473), and AKT in NC or shPTEN cells at different times post-UVB (20 mJ/cm²). B, immunoblot analysis of p-p38, p38, p-AKT (serine 473), AKT, Myr-AKT (by HA), and β-actin in HaCaT cells infected with an adenoviral vector expressing constitutively active AKT (Myr-AKT) or empty vector at 1.5 h post-sham or -UVB. C, immunoblot analysis of p-p38, p38, p-AKT, AKT, PTEN, and β-actin at 6 h post-UVB in cells that have not been exposed to UVB (First, 1^st) and in cells that have been exposed to UVB (20 mJ/cm²) once one week before. D, immunoblot analysis of p-p38, p38, p-AKT, AKT, PTEN, and β-actin in shPTEN cells treated with vehicle or LY (10 μM) at 1.5 h post-UVB, in comparison with NC cells. E, Immunoblot analysis of XPC and β-actin in HaCaT cells treated with vehicle or SB203580 (SB, 10 μM). F, enzyme-linked immunosorbent assay (ELISA) of percent (%) repair of CPDs from cells in E at different times post-UVB (20 mJ/cm²). G, immunoblot analysis of PTEN, p-AKT, AKT, p-p38, p38, XPC, and GAPDH in normal human skin and human SCCs.