Skp2 targeting suppresses tumorigenesis by Arf-p53-independent cellular senescence

Abstract

Cellular senescence has been recently shown to have an important role in opposing tumour initiation and promotion. Senescence induced by oncogenes or by loss of tumour suppressor genes is thought to critically depend on induction of the p19(Arf)-p53 pathway. The Skp2 E3-ubiquitin ligase can act as a proto-oncogene and its aberrant overexpression is frequently observed in human cancers. Here we show that although Skp2 inactivation on its own does not induce cellular senescence, aberrant proto-oncogenic signals as well as inactivation of tumour suppressor genes do trigger a potent, tumour-suppressive senescence response in mice and cells devoid of Skp2. Notably, Skp2 inactivation and oncogenic-stress-driven senescence neither elicit activation of the p19(Arf)-p53 pathway nor DNA damage, but instead depend on Atf4, p27 and p21. We further demonstrate that genetic Skp2 inactivation evokes cellular senescence even in oncogenic conditions in which the p19(Arf)-p53 response is impaired, whereas a Skp2-SCF complex inhibitor can trigger cellular senescence in p53/Pten-deficient cells and tumour regression in preclinical studies. Our findings therefore provide proof-of-principle evidence that pharmacological inhibition of Skp2 may represent a general approach for cancer prevention and therapy.

Figure 1. Skp2 loss triggers a new senescence response in MEFs in the context of Pten inactivation and Arf deficiency by a p19^Arf–p53-independent pathway

a, Primary MEFs at passage 5 from various mouse embryos were plated for senescence assay. WT, wild type. b, Cell lysates were collected from primary MEFs of various genotypes of mouse embryos for western blot analysis. The lysates from wild-type MEFs treated with γ-irradiation for 60 min served as a positive control for p53. c, Primary Pten^+/− Skp2^−/− MEFs infected with retroviruses expressing various control shRNA (shRNA-ctrl), p53 shRNA (shp53), or dominant-negative p53 (p53-DN) were plated for senescence assay and western blot analysis. d, Primary MEFs at passage 5 from various genotypes of mouse embryos were plated for senescence assay. Results are presented as mean ± s.d. from a representative experiment performed in triplicate. ***P < 0.001 using two-tailed Student's t-test, n = 3.

Figure 2. Upregulation of p27, p21 and ATF4 drives cellular senescence in Pten^+/− Skp2^−/− and Arf^−/− Skp2^−/− MEFs

a, b, Cell lysates were collected from primary MEFs of various genotypes of mouse embryos for western blot analysis. c, d, Pten^+/− Skp2^−/− MEFs at passage 2 infected with lentiviruses expressing green fluorescent protein (GFP) shRNA (shGFP) or p27 shRNA (shp27-1 and -2) were plated for western blot analysis (c) and senescence assay (d). Results are mean ± s.d. from a representative experiment performed in triplicate. e, f, Pten^+/− Skp2^−/− MEFs at passage 2 infected with lentiviruses expressing GFP shRNA or Atf4 shRNA were plated for western blot analysis (e) and senescence assay (f). g, h, Pten^+/− Skp2^−/− MEFs at passage 2 infected with lentiviruses expressing GFP shRNA or p21 shRNA were plated for western blot analysis (g) and senescence assay (h).

Figure 3. Skp2 deficiency restricts tumorigenesis after Pten inactivation by inducing cellular senescence in vivo

a, Kaplan–Meier plot analysis of cumulative survival of indicated mouse genotypes. b, Top, adrenal tissues from various mouse genotypes (10–12 months) were analysed for tumorigenesis; bottom, lymphoid tissues within the neck were obtained and weighed from female mice of various genotypes (around 5 months). **P < 0.01 using two-tailed Student's t-test, n = 6. c, Histological analysis of adrenal tissues from 12-month-old, Pten^+/− and Pten^+/− Skp2^−/− mice. Pten^+/− mice developed adrenal tumour (pheochromocytoma), which was profoundly inhibited in Pten^+/− Skp2^−/− mice. d, Senescence analysis of lymphoid tissue from 5-month-old Pten^+/− and Pten^+/− Skp2^−/− mice. e, Lymphoid tissues from 5-month-old mice of the indicated genotypes were obtained for p27 immunohistochemistry. f, Quantification of Ki67 staining of lymphoid tissues from 5-month-old mice. Results are mean ± s.d. **P < 0.01 using two-tailed Student's t-test, n = 3.

Figure 4. Skp2 inactivation restricts tumorigenesis upon Arf deficiency

a, Kaplan–Meier plot analysis of cumulative survival of Arf^−/− and Arf^−/− Skp2^−/− mice. b, A cohort of wild-type, Arf^−/− and Arf^−/− Skp2^−/− mice were analysed for tumorigenesis within a 1-year period. Arf ^−/− Skp2^−/− mice did not develop any tumour up to 1 year observation. Nine out of twenty-seven Arf ^−/− mice developed either sarcoma or lymphomas, whereas none of 12 Arf ^−/− Skp2^−/− mice developed tumours. The statistic was analysed by chi-squared test χ². c, Histopathological analysis of skin tissues from Arf^−/− and Arf^−/− Skp2^−/− mice at 1 year old. Arrow indicates sarcoma. d, Histopathological analysis of lymphoid tissues lymphoid tissues from Arf^−/− and Arf^−/− Skp2^−/− mice at 1 year of age. The arrow indicates lymphoma. The original magnification for (c) and (d) is 40X.

Figure 5. Skp2 deficiency restricts prostate cancer development by triggering cellular senescence in vivo.

a, Biopsy of anterior prostate (AP) tumours at 24 weeks from various genotypes of mice and their actual sizes and weights. Results are mean ± s.d. *P < 0.05 using two-tailed Student's t-test, n = 5. b, c, Senescence analysis of anterior prostate from Pten^pc−/− and Pten^pc−/− Skp2^−/− mice aged 3 months (b) or 15 months (c). A representative section from three mice is presented for each genotype. The original magnification for (b) and (c) is 40X and 20X, respectively. d, PC3 cells were treated with vehicle or 0.1 μM MLN4924 for 4 days and collected for cellular senescence assay. e, Nude mice bearing PC3 xenograft tumours (around 300 mm³) were treated with vehicle or MLN492, and tumour weight was measured. f, Working model for tumour-suppressive cellular senescence driven by oncogenic insults and Skp2 deficiency.