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, 25 (6), 1050-1062

Oncogene-induced Senescence Mediated by c-Myc Requires USP10 Dependent Deubiquitination and Stabilization of p14ARF

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Oncogene-induced Senescence Mediated by c-Myc Requires USP10 Dependent Deubiquitination and Stabilization of p14ARF

Aram Ko et al. Cell Death Differ.

Abstract

Oncogene-induced senescence (OIS) is a critical tumor-suppressor mechanism, which prevents hyper-proliferation and transformation of cells. c-Myc promotes OIS through the transcriptional activation of p14ARF followed by p53 activation. Although the oncogene-mediated transcriptional regulation of p14ARF has been well addressed, the post-translational modification of p14ARF regulated by oncogenic stress has yet to be investigated. Here, we found that c-Myc increased p14ARF protein stability by inducing the transcription of ubiquitin-specific protease 10 (USP10). USP10, in turn, mediated the deubiquitination of p14ARF, preventing its proteasome-dependent degradation. USP10-null mouse embryonic fibroblasts and human primary cells depleted of USP10 bypassed c-Myc-induced senescence via the destabilization of p14ARF, and these cells displayed accelerated hyper-proliferation and transformation. Clinically the c-Myc-USP10-p14ARF axis was disrupted in non-small cell lung cancer patients, resulting in significantly worse overall survival. Our studies indicate that USP10 induced by c-Myc has a crucial role in OIS by maintaining the stability of key tumor suppressor p14ARF.

Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Fig. 1
Fig. 1
c-Myc increases p14ARF protein stability through the induction of USP10. a HFF cells stably expressing c-Myc by lentivirus injection were treated with CHX (100 ng/ml) as indicated, and cell lysates were detected with p14ARF, p53, c-Myc, and actin antibodies. Graphs indicate the protein levels of p14ARF and p53 quantified using the ImageJ program. b HFF cells were infected with lentiviral c-Myc and treated with 20 μM MG132 for 3 h. Cell lysates were immunoblotted with c-Myc, p14ARF and actin antibodies. The graph indicates the average of the protein levels of p14ARF quantified using the ImageJ program. Error bars indicate 95% confidence intervals. Three independent experiments were performed. c A microarray was conducted using HFF cells stably expressing mock vector and c-Myc. Graphs indicate the fold change in mRNA levels of USPs. This experiment was conducted in triplicate. d H1299 cells were transfected with 20 nM USP1, 10, 13, and 31 siRNAs for 72 h. Cell lysates were immunoblotted with p14ARF and actin antibodies. e HFF cells were injected with lentiviral c-Myc and shUSP10 and treated with CHX as indicated, followed by immunoblotting using specific antibodies. Graphs indicate the protein levels of p14ARF and p53 quantified using the ImageJ program. f HFF cells were injected with lentiviral c-Myc. Cell lysates were immunoblotted with specific antibodies, and mRNA levels were analyzed by Q-RT-PCR using p14ARF- and USP10-specific primers. Three independent experiments were performed
Fig. 2
Fig. 2
USP10 is a transcriptional target of c-Myc. a Diagram of the USP10 promoter. There are five c-Myc binding elements, E-box sequences, upstream of the transcription start site of USP10. b A ChIP assay was performed using c-Myc antibodies in HFF and IMR90 cells stably expressing c-Myc. Relative enrichment was determined by quantifying PCR products obtained from three experiments. c 0.5 μg USP10 luciferase reporter constructs and 0.3–0.5 μg c-Myc-expressing plasmid were transfected into HFF cells as indicated. Luciferase activities of each reporter were analyzed by the luciferase assay. Cell lysates were immunoblotted with specific antibodies. This experiment was conducted in triplicate. d Luciferase activities of 0.5 μg USP10 luciferase reporter constructs, which have E-box sequence mutations of CAATTG, were analyzed using a luciferase assay. This experiment was conducted in triplicate
Fig. 3
Fig. 3
USP10 interacts with and stabilizes p14ARF. a H1299 cells were transfected with plasmids expressing p14ARF (0.05 μg) and USP10 (0.5 μg). Cell lysates were evaluated using specific antibodies. b H1299 cells were transfected with plasmids expressing p14ARF (0.05 μg) and USP10 (0.5 μg) and were treated with CHX as indicated. p14ARF and USP10 were detected using specific antibodies. c H1299 cells were transfected with plasmids expressing USP10 (1.9 μg). Cell lysates were detected using specific antibodies. d H1299 cells were transfected with plasmids expressing USP10 (1.9 μg) and treated with CHX as indicated. p14ARF and USP10 were detected using specific antibodies. e H1299 cell were transfected with 30 nM USP10 siRNAs for 72 h. Protein levels of p14ARF and USP10 were detected using specific antibodies, and mRNA levels were analyzed by Q-RT-PCR. Three independent experiments were performed. f H1299 cell were transfected with 30 nM USP10 siRNA #3 for 72 h and treated with CHX as indicated. Cell lysates were immunoblotted with specific antibodies. g, h Cells were transfected with plasmids expressing p14ARF (3 μg) and USP10 (3 μg) as indicated. Cell lysates were immunoprecipitated with HA or FLAG antibodies. p14ARF and USP10 were detected using specific antibodies. i, j Cell lysates were immunoprecipitated with p14ARF or USP10 antibodies. k Purified GST/USP10 and in vitro-translated p14ARF were incubated at 37 °C (input) followed by pulldown (PD) using glutathione-Sepharose. The precipitated samples were detected using USP10 and p14ARF antibodies
Fig. 4
Fig. 4
USP10 functions as a DUB of p14ARF. a, b H1299 cells were transfected with 0.8 μg USP10- and 0.1 μg p14ARF-expressing plasmids and treated with 20 μM MG132 for 3 h. Cell lysates were detected with specific antibodies. c Cells were transfected with plasmids expressing USP10 WT (3 μg), USP10 C424A (3 μg), and p14ARF (3 μg) as indicated, and cell lysates were immunoprecipitated with FLAG antibodies. d, f Cells were transfected with plasmids expressing USP10 WT (0.8 μg), USP10 C424A (0.8 μg), MKRN1 (0.8 μg), and p14ARF (0.1 μg) as indicated. Cell lysates were immunoblotted with specific antibodies. g Cells were transfected with plasmids expressing USP10 (4 μg), MKRN1 (4 μg), p14ARF (2 μg), and Ub (2 μg) as indicated. Cell lysates were immunoprecipitated with HA antibodies under denaturation conditions. h Cells were transfected with 30 nM control siRNA or USP10 siRNA for 72 h followed by transfection with HA/Ub (10 μg). Cell lysates were immunoprecipitated with p14ARF antibodies under denaturation conditions
Fig. 5
Fig. 5
USP10 is required for c-Myc-induced cellular senescence. a HFF cells were infected with lentiviral c-Myc and shUSP10. Cell lysates were immunoblotted with specific antibodies. b Stable cells were stained for β-galactosidase activity, and β-gal-positive cells from three independent experiments were counted. c Stable cells were seeded in a soft agar matrix and cultured for 8 days. Anchorage-independent cell growth was analyzed using the MTT assay. d HFF and IMR90 cells were injected with lentiviral USP10 and shp14ARF. Cell lysates were immunoblotted with specific antibodies. e Cells were stained for β-galactosidase activity. β-gal-positive cells from three independent experiments were counted. f The same number of cells was seeded and counted for 7 days. Error bars indicate 95% confidence intervals. Three independent experiments were performed
Fig. 6
Fig. 6
USP10-KO MEFs escape from c-Myc-induced cellular senescence. a WT and USP10-KO MEFs were immunoblotted with specific antibodies. b MEFs were stained for β-galactosidase activity, and β-gal-positive cells from three independent experiments were counted. c The same number of cells was seeded and counted for 5 days. Error bars indicate 95% confidence intervals. Three independent experiments were performed. d MEFs were infected with retroviral c-Myc. Cell lysates were immunoblotted with specific antibodies. e Cells were stained for β-galactosidase activity, and β-gal-positive cells from three independent experiments were counted. f The same number of cells was seeded and counted for 5 days. Error bars indicate 95% confidence intervals. Three independent experiments were performed. g Cells were seeded in a soft agar matrix and cultured for 8 days. Colonized cells from three independent experiments were counted
Fig. 7
Fig. 7
The functional axis of c-Myc-USP10-p14ARF in human non-small cell lung cancer specimens is disrupted. a Representative images of immunohistochemical staining of c-Myc, USP10, and p14ARF in non-small cell lung cancer tissues. High magnification images are shown in the inset. The scale bar is 100 µm. b Overall survival curves for non-small cell lung cancer patients according to combined markers groups. Patients with combined c-Myc+/USP10-, c-Myc+/p14ARF-, USP10-/p14ARF-, and c-Myc+/USP10-/p14ARF- expression showed significantly worse overall survival (p = 0.008, p = 0.021, p = 0.035 and p = 0.021, respectively) than patients with combined c-Myc-/USP10+, c-Myc-/p14ARF+, USP10+/p14ARF+, and c-Myc-/USP10+/p14ARF+ expression. c Proposed model. Oncogenic c-Myc induces USP10 transcription to strengthen the fail-safe program by deubiquitination-dependent stabilization of p14ARF and p53, which prevents immortalization and tumorigenesis in normal cells

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