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USP15 stabilizes MDM2 to mediate cancer-cell survival and inhibit antitumor T cell responses

Nature Immunology volume 15, pages 562–570 (2014)Cite this article

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Abstract

Deubiquitinases (DUBs) are a new class of drug targets, although the physiological function of only few DUBs has been characterized. Here we identified the DUB USP15 as a crucial negative regulator of T cell activation. USP15 stabilized the E3 ubiquitin ligase MDM2, which in turn negatively regulated T cell activation by targeting the degradation of the transcription factor NFATc2. USP15 deficiency promoted T cell activation in vitro and enhanced T cell responses to bacterial infection and tumor challenge in vivo. USP15 also stabilized MDM2 in cancer cells and regulated p53 function and cancer-cell survival. Our results suggest that inhibition of USP15 may both induce tumor cell apoptosis and boost antitumor T cell responses.

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Figure 1: Negative regulation of naive CD4+ T cell activation by USP15.
Figure 2: USP15 deficiency promoted T cell responses to L. monocytogenes infection.
Figure 3: USP15 deficiency inhibited ubiquitin-dependent degradation of NFATc2 in activated T cells.
Figure 4: USP15 mediated deubiquitination and stabilization of MDM2.
Figure 5: MDM2 negatively regulated NFATc2 activation and cytokine induction in T cells in a p53-independent manner.
Figure 6: USP15 stabilized MDM2 and negatively regulated p53-dependent gene expression and apoptosis in cancer cells.
Figure 7: USP15-mediated MDM2 stabilization promoted cancer cell survival and inhibited antitumor T cell responses.

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Acknowledgements

We thank W. Gu for expression vectors, B. Vogelstein for HCT116 derivative cell lines, S. Hao for recombinant L. monocytogenesis, Q. Yi for B16 cell line, L.A. Donehower for Trp53−/− mice, and the personnel from the US National Institutes of Health or National Cancer Insitute–supported resources (flow cytometry, DNA analysis and animal facilities) under award number P30CA016672 at The MD Anderson Cancer Center. This study was supported by grants from US National Institutes of Health (AI057555, AI064639, GM84459 and AI104519 to S.-C.S. and AI098099 to S.S.W.) and partially supported by a Sister Institution Network Fund and a seed fund from the Center for Inflammation and Cancer at the MD Anderson Cancer Center.

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Authors and Affiliations

  1. Department of Immunology, The University of Texas MD Anderson Cancer Center, Houston,, Texas, USA

    Qiang Zou, Jin Jin, Hongbo Hu, Haiyan S Li, Simona Romano, Yichuan Xiao, Mako Nakaya, Xiaofei Zhou, Xuhong Cheng, Chengming Zhu, Stephanie S Watowich, Stephen E Ullrich & Shao-Cong Sun

  2. Department of Molecular Medicine and Medical Biotechnologies, Federico II University, Naples, Italy

    Simona Romano

  3. Department of Genetics, The University of Texas MD Anderson Cancer Center, Houston,, Texas, USA

    Peirong Yang & Guillermina Lozano

  4. The University of Texas Graduate School of Biomedical Sciences, Houston, Texas, USA

    Guillermina Lozano, Chengming Zhu, Stephanie S Watowich, Stephen E Ullrich & Shao-Cong Sun

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Contributions

Q.Z. designed and performed the experiments, prepared the figures and wrote part of the manuscript; J.J., H.H., H.S.L., S.R., Y.X., M.N., X.Z., X.C., P.Y. and C.Z. contributed to the performance of the experiments; S.S.W. and G.L. were involved in supervision of H.S.L. and P.Y., respectively; S.E.U. was involved in collaboration on cancer model studies, and S.-C.S. supervised the work and wrote the manuscript.

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Correspondence to Shao-Cong Sun.

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Integrated supplementary information

Supplementary Figure 1 Generation and initial characterization of Usp15−/− mice.

(a) Schematic picture of Usp15 gene targeting using the OmniBank retroviral gene-trapping technique. (b-d) Genotyping PCR analysis of tail DNAs (b), RT-PCR analysis of the indicated regions of Usp15 cDNA in T cells (c), and USP15 immunoblot using T cells (d) of WT (+/+), heterozygous (+/−), and Usp15−/− (−/−) mice. (e) Survival curve of WT and Usp15−/− mice. (f) Flow cytometric analysis of thymocytes from WT and Usp15−/− mice (6 wk old), showing the percentage of CD4−CD8− double negative, CD4+CD8+ double positive, and the CD4+ and CD8+ single positive populations (n=5; showing a representative plot). (g) Flow cytometric analysis of the frequency of CD3+ T cells and the CD19+ B cells in the splenocytes of WT and Usp15−/− mice (8-12 wk old) (n=5; showing a representative plot). (h,i) Flow cytometric analysis of the frequency of naïve (CD44loCD62Lhi) and memory (CD44hiCD62Llo) populations in the splenic CD4+ T cells from WT or Usp15−/− mice of 3 (h) or 10 (i) months of age (n=5; showing a representative plot). Data are representative of three (f-i) independent experiments.

Supplementary Figure 2 Effect of USP15 deficiency on CD4+ T cell activation and differentiation.

(a,b) Rag1−/− mice were adoptively transferred with WT or Usp15−/− bone marrow cells transduced with a GFP-expressing lentiviral vector (EV) or the same vector encoding USP15 or a catalytically inactive USP15 mutant (C298A). GFP+ thymocytes (a) and splenic naïve CD4+ T cells (b) were sorted from the indicated chimeric mice and subjected to USP15 IB (a) and intracellular IFN-γ staining following stimulation with anti-CD3 plus anti-CD28 for 72 h (b) (n=3; showing a representative plot). (c-e) Naïve CD4+ T cells from WT or Usp15−/− (KO) mice were stimulated under standard TH0, TH1, TH2, TH17 or iTreg conditions and harvested on day 5 for qRT-PCR (c), proliferation (d), and apoptosis assays (e) (n=3). (f) Flow cytometric analysis of Treg cells differentiated for 5 days from WT or Usp15-deficient naive CD4+ T cells under Treg cell conditions with different doses of TGF-β in the presence (+) or absence (−) of an anti-IFN-γ blocking antibody. (g) Flow cytometric analysis of the frequency of Foxp3+ Treg cells among CD3+CD4+CD8- cells in the indicated lymphoid organs of WT or Usp15−/− mice (5 wk old). Data are presented as a representative plot (left) and a summary graph (right) (n=6). All summary graphs are mean ± s.e.m and data are representative of three independent experiments. * P <0.05.

Supplementary Figure 3 USP15 deficiency enhanced T cell response to listeria infection.

(a,b) Flow cytometric analyses of the number of IFN-γ-producing CD4+ T cells (a) and the IFN-γ MFI in the IFN-γ+CD4+ T cells (b) from the spleen of wild-type and Usp15−/− mice infected with L. monocytogenes for 6 days (n=5). (c-e) Tcrb−/−Tcrd−/− mice were reconstituted with 5 × 106 wild-type or Usp15−/− (KO) naïve CD4+ T cells in the presence of equal numbers of wild-type CD8+ T cells. After 4 days, the recipient mice were sacrificed for visualizing spleen size (c, n=3), flow cytometric analysis of splenic CD4+ T cells (d, n=5), and ELISA of serum IFN-γ concentration (e, n=5). (f, g) Spleen size (f, n=3) and flow cytometric analysis of the frequency of CD11b+ monocytes and CD11c+ dendritic cells in splenocytes (g, n=5) of the reconstituted Tcrb−/−Tcrd−/− mice, infected i.v. with 5 × 104 CFU of L. monocytogenes for 4 days. wild-type or Usp15−/−, Tcrb−/−Tcrd−/− mice reconstituted with wild-type or Usp15−/− naïve CD4+ T cells (c-g). (h,i) B6.SJL mice, adoptively transferred with wild-type OT-II or Usp15−/− OT-II naïve CD4+ T cells, were either uninfected (UI) or infected for 6 days with OVA-expressing L. monocytogenes (LM) and subjected to flow cytometric analyses of the number of the total transferred T cells (CD45.2+) (h) and Treg cells (Foxp3+CD45.2+) (i) in the spleen (n=3 for uninfected and 5 for LM-infected mice). Wild-type OT-II or KO OT-II, B6.SJL mice adoptively transferred with wild-type OT-II or Usp15−/− OT-II naïve CD4+ T cells (h, i). UI, uninfected. LM, L. monocytogenes infected. Data are presented as mean ± s.e.m. and representative of at least three independent experiments. * P<0.05; ** P<0.01.

Supplementary Figure 4 USP15 was dispensable for TCR-proximal signaling and TGF-β signaling in T cells.

(a,b) IB analysis of the indicated phosphorylated (p-) and total proteins in the whole-cell lysates of naive CD4+ T cells stimulated with anti-CD3 and anti-CD28 antibodies using a crosslinking protocol. (c,d) wild-type or Usp15−/− naïve CD4+ T cells were stimulated with anti-CD3 plus anti-CD28 for 24 h, starved for 24 h, and then stimulated with TGF-β for the indicated time periods. Cells were subjected to IB (c) or qRT-PCR analysis (d) (n=3). (e,f) qRT-PCR analysis of Il2 and Ifng mRNA (e) and ELISA of secreted IL-2 and IFN-γ (f) in naïve CD4+ T cells stimulated for 24 h (e) or 48 h (f) (n=3). (g) IB analysis using cytoplasmic (CE) and nuclear (NE) extracts of naive CD4+ T cells stimulated with anti-CD3 plus anti-CD28 in the presence (+) or absence (−) of TGF-β. (h) IB analysis of NFATc2 in EL4 T cells infected with a retroviral vector (EV) or the same vector encoding NFATc2. (i,j) Intracellular staining of IFN-γ (i, n=5; showing a representative plot) and ELISA of IL-2 and IFN-γ (j, n=3) in the EL4 cells described in h, stimulated as indicated. (k) qRT-PCR analysis of Nfatc2 mRNA relative level (normalized to the control Actb) in wild-type or Usp15−/− naïve CD4+ T cells, stimulated as indicated (n=3). Data in graphs are presented as mean ± s.e.m, and representative of at least three independent experiments. * P<0.05.

Supplementary Figure 5 MDM2 associated with USP15 and regulated TCR/CD28-stimulated NFATc2 activation and cytokine induction in a p53-independent manner.

(a) MDM2-USP15 co-IP (upper) and direct IB (lower) assays using cytoplasmic (CE) and nuclear (NE) extracts of wild-type naïve CD4+ T cells, incubated for 2 h with MG132 either in the absence (−) or presence of anti-CD3 plus anti-CD28. (b) IB analysis using cytoplasmic (CE) and nuclear (NE) extracts of untreated (UT) wild-type or Usp15−/− naïve CD4+ T cells. (c) IB analysis using cytoplasmic (CE) and nuclear (NE) extracts of wild-type naive CD4+ T cells incubated with (+) or without (−) anti-CD3 plus anti-CD28 for 3 h in the presence of MG132 during the last 2 h. (d) NFATc2 ubiquitination (upper) and IB (lower) analyses using HEK293 cells transfected with the indicated expression vectors. (e,f) IB (e) and qRT-PCR (f, n=3) analyses in naïve CD4+ T cells stimulated with anti-CD3 plus anti-CD28 in the absence (−) or presence of HLI373 or DMSO. (g-i) IB analyses using GFP+ thymocytes (g) or naïve CD4+ T cells (h) and IFN-γ ICS in stimulated GFP+ naïve CD4+ T cells (n=5; showing a representative plot) of Rag1−/− mice adoptively transferred (for 6 wk) with Usp15−/− bone marrow cells transduced with a GFP-expressing vector (EV) or the same vector encoding HA-tagged MDM2. (j) IB analysis using sorted (GFP+) wild-type or Usp15−/− naïve CD4+ T cells reconstituted, via bone marrow adoptive transfer, with a GFP-expressing vector (EV), USP15, or USP15C298A and stimulated as indicated. (k,l) qRT-PCR (k) and IB (l) analyses of wild-type naive CD4+ T cells stimulated with anti-CD3 plus anti-CD28 in the presence of DMSO or Nutlin-3 (n=3). Data in summary graphs are mean ± s.e.m, and representative of at least three independent experiments. * P<0.05.

Supplementary Figure 6 CD8+ T cells expressed lower levels of MDM2 and were not influenced by USP15 deficiency in cytokine production.

(a) IB analysis of USP15 and MDM2 protein levels in CD4+ and CD8+ T cells. A nonspecific band is indicated as NS. (b) qRT-PCR analysis of Mdm2 and Usp15 mRNA levels in CD4+ and CD8+ T cells (n=3). (c) IB analysis of NFATc2 and loading controls in the nuclear (NE) and cytoplasmic (CE) extracts of wild-type or Usp15−/− CD8+ T cells stimulated as indicated. (d,e) ELISA (d, n=3) and qRT-PCR (e, n=3) analyses using wild-type and Usp15−/− (KO) CD8+ T cells stimulated for the indicated time periods with anti-CD3 plus anti-CD28. Data are representative of three independent experiments. NS, P>0.05, **P<0.01.

Supplementary Figure 7 USP15 was abundantly expressed in cancer cells and required for MDM2 expression and cancer cell survival.

(a) IB analysis using whole-cell extracts of melanoma and colon cancer cell lines. (b) IB analysis of the indicated proteins in human primary dermal fibroblasts transduced with pGIPZ lentiviral vectors encoding either a control shRNA (C) or two different USP15 shRNAs. (c) IB analysis of the indicated proteins using WT or Usp15−/− murine B cells and thymocytes. (d,e) Control shRNA (Ctrl)-transduced or USP15 shRNA (shUSP15)-transduced A375 cells were reconstituted with either an empty vector (EV) or an RNAi-resistant USP15 expression vector. The cells were subject to IB analysis of the indicated proteins (d) or flow cytometric analysis of spontaneous apoptosis showing a representative plot (numbers in the figure panels indicate percentage of apoptotic cells) and a summary graph (e) (n=3). (f) IB analysis of the indicated proteins using HCT116 p53 WT or HCT116 p53 null (Trp53 −/−) cells infected with control (c) or USP15 shRNAs. (g) Apoptotis analyses of the HCT116 p53 WT and HCT116 p53 null (Trp53 −/−) cells (n=3). Bar graphs are mean ± s.e.m. Data are representative of three independent experiments. *P<0.05; **P<0.01.

Supplementary Figure 8 USP15 regulated the stability of MDM2 but was dispensable for TGF-β signaling in A375 and HCT116 cells.

(a) IB analysis of MDM2 in control (C) or USP15 knockdown (shRNA 1 and shRNA 2) A375 cells incubated for 4 h with10 μg/ml MG132 or the solvent control DMSO. (b) IB analysis of the indicated proteins in control (Ctrl) or USP15 knockdown (shRNA 1) cells incubated with cycloheximide (50 μg/ml) for the indicated time periods. (c) A375 and HCT116 cells were stably infected with a non-silencing control shRNA (C) or two different USP15 shRNAs. The cells were serum-starved for 24 h and stimulated with TGF-β for 1h, and whole-cell lysates were subjected to IB analysis of the indicated phosphorylated (p-) and total proteins. (d,e) qRT-PCR analysis of Pai1 and Smad7 mRNA in the indicated cells, either untreated (UT) or stimulated with TGF-β for 4h (n=3; mean ± s.e.m.). Data are representative of three independent experiments.

Supplementary Figure 9 USP15 regulated tumor growth and antitumor T cell responses.

(a) Growth curve of xenograft tumors in nude mice that were injected s.c. with HCT116 cells (1× 107 cells per mouse) stably expressing a control (Ctrl) shRNA or a USP15-specific shRNA (shRNA 1) (n=7). (b) IB analysis of the indicated proteins using lysates the xenograft tumors collected on day 21 post injection. (c) Growth curve of xenograft tumors in nude mice injected s.c. with USP15-silenced HCT116 p53 null (Trp53 −/−) cells (5 × 106 cells per mouse) (n=5). (d) IB analysis using control (Ctrl) or USP15-knockdown B16 tumor cells. (e,f) Growth curve of tumors in wild-type or Usp15−/− (KO) mice injected s.c. with a low dose (5 × 105 cells per mouse) (e) or a high dose (5 × 106 cells per mouse) (f) of the indicated B16 cells (n=5). (g,h) wild-type and Usp15−/− mice were injected s.c. with B16 melanoma cells. After 15 days of challenge, tumors were removed for ICS and flow cytometry analyses of the number of IFN-γ-producing tumor-infiltrating CD4+ (g) and the frequency of IFN-γ-producing tumor-infiltrating CD8+ (h) T cells. Data are presented as a representative plot or a summary graph of multiple mice (n=5). Summary graphs are mean ± s.e.m., and data are representative of three independent experiments. * P<0.05.

Supplementary Figure 10 USP15 deficiency promoted IFN-γ−dependent tumor suppression but did not influence Treg cell frequency.

(a,b) Flow cytometry analysis of the frequency of Foxp3+CD4+ T cells in the spleen, draining lymph node (dLN), and tumor-infiltrating lymphocytes (TIL) of day 15 B16-challenged wild-type and Usp15−/− mice. Data are presented as a representative plot (a) and a summary graph of multiple mice (n=10) (b). (c) Tumor growth curves in wild-type or Usp15−/− mice injected s.c. with B16 melanoma cells and administered i.p. with 0.5 mg of an anti-mouse IFN-γ neutralizing antibody or a rat IgG control every 3 days starting from day 1 of B16 challenge. (d) Flow cytometric analysis of splenic CD4+ T cells in Tcrb−/−Tcrd−/− mice reconstituted (for 16 days) with 5 × 106 wild-type or Usp15−/− (KO) naïve CD4+ T cells in the presence of equal numbers of wild-type CD8+ T cells (n=5). Summary graphs are mean ± s.e.m., and data are representative of three independent experiments. NS, P>0.05; * P<0.05.

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Zou, Q., Jin, J., Hu, H. et al. USP15 stabilizes MDM2 to mediate cancer-cell survival and inhibit antitumor T cell responses. Nat Immunol 15, 562–570 (2014). https://doi.org/10.1038/ni.2885

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