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Deubiquitylase OTUD3 regulates PTEN stability and suppresses tumorigenesis

Nature Cell Biology volume 17, pages 1169–1181 (2015)Cite this article

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Abstract

PTEN is one of the most frequently mutated tumour suppressors and reduction in PTEN protein stability also plays a role in tumorigenesis. Although several ubiquitin ligases for PTEN have been identified, the deubiquitylase for de-polyubiquitylation and stabilization of PTEN is less defined. Here, we report OTUD3 as a deubiquitylase of PTEN. OTUD3 interacts with, de-polyubiquitylates and stabilizes PTEN. Depletion of OTUD3 leads to the activation of Akt signalling, induction of cellular transformation and cancer metastasis. OTUD3 transgenic mice exhibit higher levels of the PTEN protein and are less prone to tumorigenesis. Reduction of OTUD3 expression, concomitant with decreased PTEN abundance, correlates with human breast cancer progression. Furthermore, we identified loss-of-function OTUD3 mutations in human cancers, which either abolish OTUD3 catalytic activity or attenuate the interaction with PTEN. These findings demonstrate that OTUD3 is an essential regulator of PTEN and that the OTUD3–PTEN signalling axis plays a critical role in tumour suppression.

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Figure 1: The deubiquitylase OTUD3 stabilizes PTEN.
Figure 2: OTUD3 de-polyubiquitylates PTEN.
Figure 3: OTUD3 regulates cytoplasmic PTEN and inhibits Akt signalling.
Figure 4: OTUD3 suppresses tumorigenesis.
Figure 5: OTUD3 transgenic mice exhibit higher PTEN expression and are less prone to tumorigenesis.
Figure 6: OTUD3 E86K mutant loses DUB function and gains oncogene function.
Figure 7: A subset of OTUD3 mutations in human cancer attenuate the ability to interact with and stabilize PTEN.
Figure 8: Positive correlation between OTUD3 and PTEN in human breast cancer.

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Acknowledgements

We thank Q. Ye (Beijing Institute of Biotechnology, Beijing, China), Z. Liu (Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China) and S. Maddika (Centre for DNA Fingerprinting and Diagnostics, Nampally, Hyderabad, India) for kindly providing materials and H. Li, P. Xie, Y. Chen, S. Wang, J. Feng, M. Lai and D. Zhao for technical assistance. This work was supported by Chinese National Basic Research Programs (2012CB910702, 2011CB910602, 2012CB910304), the Program of International S&T Cooperation (2014DFB30020), Chinese National Natural Science Foundation Projects (31330021, 31125010, 81221004) and Beijing Natural Science Foundation Project (5142020).

Author information

Author notes

  1. Lin Yuan, Yanrong Lv and Hongchang Li: These authors contributed equally to this work.

Authors and Affiliations

  1. State Key Laboratory of Proteomics, Beijing Proteome Research Center, Beijing Institute of Radiation Medicine, Collaborative Innovation Center for Cancer Medicine, Beijing 100850, China

    Lin Yuan, Hongchang Li, Shanshan Song, Yuan Zhang, Guichun Xing, Xiangzhen Kong, Fuchu He & Lingqiang Zhang

  2. Institute of Cancer Stem Cell, Dalian Medical University, Liaoning Province 116044, China

    Lin Yuan & Lingqiang Zhang

  3. Department of General Surgery, QiLu Hospital of Shandong University, Shandong Province 250012, China

    Yanrong Lv & Haidong Gao

  4. Vascular Biology Research Institute, Guangdong Pharmaceutical University, Guangdong Province 510006, China

    Lijing Wang

  5. Department of Experimental Pathology, Beijing Institute of Radiation Medicine, Beijing 100850, China

    Yang Li

  6. Institute of Basic Medical Sciences, China National Center of Biomedical Analysis, Beijing 100850, China

    Tao Zhou

  7. Key Laboratory of Systems Biology, Innovation Center for Cell Signaling Network, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China

    Daming Gao

  8. School of Life Sciences, Sichuan University, Sichuan Province 610065, China

    Zhi-Xiong Xiao

  9. Institute of Systems Biomedicine, Center for Systems Biology and Medicine of Aging, Peking University Health Science Center, Beijing 100191, China

    Yuxin Yin

  10. Department of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02115, USA

    Wenyi Wei

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Contributions

The project was conceived by L.Z. The experiments were designed by L.Z., L.Y., Y.Lv, H.L., Y.Y. and F.H. Most of the experiments were performed by L.Y., Y.Lv and H.L. The establishment and phenotype analysis of transgenic mice were contributed by H.L. The protein ubiquitylation assays, the protein interaction assays and animal experiments were contributed by S.S., Y.Z., G.X., X.K. and L.W. The breast cancer sample collection and immunohistochemical analysis were contributed by H.G., Y.Li, T.Z. and D.G. The data were analysed by L.Z., L.Y., Y.Lv, H.L., Z.-X.X., Y.Y., W.W. and F.H. The manuscript was written by L.Z., L.Y. and Y.Lv.

Corresponding author

Correspondence to Lingqiang Zhang.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 4 OTUD3 stabilizes PTEN protein.

a, Protein level analysis of PTEN in the presence or absence of overexpressed DUBs. HCT116 colon cancer cells were overexpressed with each of the Flag-tagged DUBs. b, Quantification of the PTEN protein levels relative to β-actin for Fig. 1b. n = 3 independent experiments (**, P < 0.01, Student’s t-test). c, Increasing amounts of plasmids encoding other members of OTU sub-family of DUBs were each transfected into HCT116 cells. The endogenous PTEN protein levels were examined by western blot. d, Knockdown of OTUD3 by pool siRNAs (left) or individual siRNAs (middle) or overexpression of OTUD3 (right) had no significant effect on PTEN mRNA level in MCF7 cells. n = 3 independent experiments (NS: no significance, Student’s t-test). e, MCF7 cells stably expressing the indicated shRNA were treated with cycloheximide (10 μg ml−1), and harvested at the indicated times. The left panels show immunoblots of PTEN and OTUD7A. Right panel: quantification of the PTEN protein levels relative to β-actin. n = 3 independent experiments (NS: no significance, two-way ANOVA test). f, Domain structure of OTUD3 and OTUD7A. g, Co-immunoprecipitation assays in four types of cancer cell lines to detect the endogenous interactions. The total cell lysates were immunoprecipitated with anti-OTUD3, anti-PTEN or control IgG as indicated. Both the lysates and the immunoprecipitates were subjected to western blot analysis. h, GST pull-down assays were performed to map the domain of OTUD3 required for interaction with PTEN. i, GST pull-down assays were performed to map the domain of PTEN required for interaction with OTUD3. All panels are representative results of three or more independent experiments. Statistics source data can be found in Supplementary Table 4. Uncropped images of blots are shown in Supplementary Fig. 9.

Supplementary Figure 5 OTUD3 is a more potent regulator of PTEN than USP13, and HAUSP has no significant effect on PTEN protein level.

a, Increasing amounts of Flag-USP13 were transfected into MCF7 (left) and HEK293T (right) cells. After 48 h, PTEN protein levels were detected. b, Co-immunoprecipitation of endogenous PTEN, USP13 and OTUD3 in MCF7 cells. Protein extracts were immunoprecipitated with antibody against PTEN, USP13 or OTUD3, followed by immunoblotting with antibodies indicated. c, Increasing amounts of shRNA-USP13 were transfected into MCF7 cells. After 72 h, PTEN protein levels were detected. d, Protein level analysis of PTEN in the presence of overexpressed USP13 or OTUD3. MCF7 cells were overexpressed with Myc-USP13 or Flag-OTUD3. e, Protein level analysis of PTEN in MCF7 cells stably expressing the indicated shRNA. f, MCF7 cells were transfected with a constant amount of shRNA-USP13 and increasing amounts of Myc-OTUD3 (left), or a constant amount of shRNA-OTUD3 and increasing amounts of Myc-USP13 (right). After 48 h, PTEN protein levels were detected. g, HCT116 cells transfected with the indicated constructs were treated with MG132 for 8 h before harvest. PTEN was immunoprecipitated with anti-PTEN and immunoblotted with anti-HA. h, siRNA-resistant OTUD3 was introduced into the HCT116 cells together with the OTUD3 siRNA. PTEN ubiquitination was measured. i, The PTEN ubiquitination linkage was analysed in HCT116 cells transfected with OTUD3and the indicated ubiquitin WT or K48-only plasmids. j, Increasing amounts of HAUSP plasmids were transfected into the MCF7 cells and the lysates were subjected to western blot analysis. k, Knockdown of HAUSP by individual siRNAs had no significant effect on PTEN protein level but downregulated MDM2 protein level (positive) in MCF7 cells. l, Effectiveness of HAUSP knockdown by individual siRNAs was measured by quantitative RT-PCR. Data are shown as mean ± s.d. n = 3 independent experiments. (**, P < 0.01, Student’s t-test). All panels are representative results of three or more independent experiments. Statistics source data can be found in Supplementary Table 4. Uncropped images of blots are shown in Supplementary Fig. 9.

Supplementary Figure 6 OTUD3 negatively regulates Akt signaling in a PTEN-dependent manner.

a,b, Quantitative RT-PCR analysis of indicated gene mRNA level in MCF7 (PTEN wild-type) (a) or BT549 (PTEN-null) (b) cells stably expressing the shRNA-Control or shOTUD3#1. Data were analysed using two-tailed unpaired Student’s t-test. Data are shown as mean ± s.d. n = 3 independent experiments. *: P < 0.05,**: P < 0.01, NS: no significance. c, Quantitative RT-PCR analysis of VEGF gene mRNA level in MCF7 and BT549 cells stably expressing shRNA-Control or shOTUD3 #1. Data were analysed using two-tailed unpaired Student’s t-test. Data are shown as mean ± s.d. n = 3 independent experiments. d,e, OTUD3 shRNA-transduced MCF7 or BT549 cells were serum-starved and treated with EGF for various times. Quantification of the phosphorylation levels of Akt, p38 and Erk protein relative to total Akt, p38 and Erk protein levels for Fig. 3f and g. Data are shown as mean ± s.d. n = 3 independent experiments (two-way ANOVA test). Statistics source data can be found in Supplementary Table 4.

Supplementary Figure 7 OTUD3 inhibits cell proliferation, promotes cell apoptosis, and suppresses cell migration.

a, Anchorage-independent growth of MCF7 cells stably expressing the indicated shRNA on soft agar. Viable colonies after 3 weeks were counted and the data from n = 3 independent experiments were presented (mean ± s.d.). **: P < 0.01, Student’s t-test. b, MCF7 cells stably expressing control shRNA, OTUD3 shRNA or PTEN shRNA were transfected with either OTUD3 or PTEN as indicated. Cells were tested for growth in colony assay. Colonies after one week were counted and the data (mean ± s.d.) from n = 3 independent experiments were presented (**: P < 0.01, NS: no significance, Student’s t-test, compared with cells expressing control shRNA;). c, MCF7 cells stably expressing control shRNA, OTUD3 shRNA or PTEN shRNA were seeded and cell proliferation was measured by MTS assay for 5 days. Data are shown as mean ± s.d. n = 3 independent experiments (**: P < 0.01, two-way ANOVA test). d, The indicated HCT116 cells were treated with cisplatin or DMSO to induce apoptosis. The percentage of apoptotic cells was measured by Annexin-V staining. Data are shown as mean ± s.d. n = 3 independent experiments (*: P < 0.05; **: P < 0.01, Student’s t-test). e, The cellular morphology was analysed in MCF7 cells stably expressing the indicated shRNA and/or transfected with the OTUD3 or PTEN plasmids. Cells were fixed and stained for F-actin (red). Nuclei were stained with DAPI (blue). Scale bar, 20 μm. f, Box plots showed the distribution of the size (largest in each section) of metastasis per section. Data were analysed using Mann–Whitney test (n = 50 fields from 10 mice, p = 0.0025). g, Immunoblotting of OTUD3, PTEN, p-Akt, Akt, F-actin and β-actin in lysates of primary tumors and metastatic livers from mice injected with the stable shRNA-control or shRNA-OTUD3 MCF7 cells. h, Pie charts for mice died by liver metastasis. Panel g is representative results of 3 independent experiments. Statistics source data can be found in Supplementary Table 4. Uncropped images of blots are shown in Supplementary Fig. 9.

Supplementary Figure 8 The possible synergy between OTUD3 and USP13, and the phenotype of OTUD3 transgenic mice.

a, Akt phosphorylation was analysed in MCF7 cells stably expressing the indicated shRNA. b–d, The MCF7 cells expressing OTUD3 WT or mutants were measured for cell proliferation (b, two-way ANOVA test), anchorage-independent growth in soft agar (c, Student’s t-test) and transwell migration assay (d, Student’s t-test). Data are shown as mean ± s.d. n = 3 independent experiments. **: P < 0.01, *: P < 0.05. e, Representative bright-field imaging of the tumors. MCF7cells stably expressing indicated shRNA were implanted into a subcutaneous site in the skin on one flank of athymic nude mice (n = 8). On 4 weeks, mice receiving transplants of indicated cells were sacrificed. Scale bar, 1 cm. f, Whisker plots showed the distribution of the tumor weights (n = 8 tumors from 8 mice). Data were shown as mean ± s.d. and analysed using Kruskal-Wallis test. g, WT and TG MEFs were serum-starved and treated with EGF for various times. Quantification of the phosphorylation levels of Akt, p38 and Erk protein relative to total Akt, p38 and Erk protein levels for Fig. 5i. Data are shown as mean ± s.d. n = 3 independent experiments (two-way ANOVA test). h, The mating strategy of OTUD3 TG mice crossing with MMTV-PyMT mice. Panels a,g are representative results of 3 or more independent experiments. Statistics source data can be found in Supplementary Table 4. Uncropped images of blots are shown in Supplementary Fig. 9.

Supplementary Figure 9 OTUD3 E86K mutation in OTU domain leads to PTEN protein destabilization and increased tumorigenesis.

a, Sequencing the region encoding OTUD3 OTU domain from 50 cases of Chinese breast cancer patients. Breast cancer tissues (n = 50) and the corresponding adjacent tissues (n = 50) were collected and subjected to RNA extraction and reverse transcription. The cDNAs were then sequenced. If the original sequencing results displayed heterozygous peaks, the corresponding cDNAs were ligated with pGEM-T vector (Promega) and transformed into the competent bacteria. Then the colonies were sent out for sequencing. b, The mutation sites of OTUD3 in domain structure. Amino acid sequence alignment spanning OTUD3 R79 and E86 across species. c, Half-life of PTEN in the presence or absence of ectopic OTUD3 mutants. Quantification of the PTEN protein levels relative to β-actin for Fig. 6d (**: P < 0.01, two-way ANOVA test). Data are depicted as bar graphs with mean ± s.d. n = 3 independent experiments. Statistics source data can be found in Supplementary Table 4.

Supplementary Figure 10 OTUD3 expression is reduced in human breast cancer, which has no correlation with estrogen receptor, progesterone receptor or HER-2 expression.

a, Relative OTUD3 mRNA level in breast cancer tissues and matched normal control from 30 subjects. Data were analysed using Chi-square test. b–d, Whisker plots show the OTUD3 mRNA level in the cancer tissues classified into negative and positive groups based on estrogen receptor (b), progesterone receptor (c) or HER-2 (d) expression from 50 subjects. Data were analysed using Mann–Whitney test. e, Representative images from immunohistochemical staining of OTUD3, PTEN and p-Akt(S473) in three serial sections of the same tumor and matched adjacent tissue. The boxed areas in the left images were magnified on the right. A, adjacent tissue; C, carcinoma. Scale bar, 50 μm.

Supplementary Figure 11 OTUD3 and USP13 exhibit a synergy effect on PTEN expression in breast cancer samples.

a,b, Potential synergy between OTUD3 and USP13 for PTEN protein levels. Serial sections of tissue arrays 68 patient breast specimens were subjected to immunohistochemistry with anti-OTUD3, anti-USP13 and anti-PTEN antibodies, respectively, and visualized by the DAB staining before imaging. Scale bar represents 50 μm. Representative images were shown in (a), and the summary of the IHC results was listed in (b). P < 0.001, calculated by both Chi-square and χ2 test. Scale bar, 50 μm. c, Regression analysis comparing HAUSP and PTEN expression in breast cancer tissues. n = 37. d, Proposed working model of OTUD3 on PTEN stability control. PTEN is a phosphatase that catalyses the conversion of the lipid second messenger PtdIns(3,4,5)P3 to PtdIns(4,5)P2 and inhibits PI3K-Akt signaling. PTEN protein is relatively stable. This study identifies the deubiquitinase OTUD3 catalyses the removal of poly-ubiquitin chain of PTEN in the cytoplasm. OTUD3 reverses the poly-ubiquitination of PTEN by E3 ubiquitin ligases (such as Nedd4-1, WWP2, XIAP and CHIP) and prevents PTEN degradation by 26S proteasome. On the other hand, a previous study (ref. 22) identified that the deubiquitinase HAUSP is mainly localized in the nucleus and catalyses the removal of mono-ubiquitination of PTEN. HAUSP regulates the PTEN cytoplasm-nucleus shuttling but has no effects on PTEN stability.

Supplementary Table 1 OTUD3 mutations in human cancer.
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Supplementary Table 2 Primers for OTUD3 mutation selection in human breast cancer.
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Supplementary Table 3 Primers for qPCR analysis.
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Yuan, L., Lv, Y., Li, H. et al. Deubiquitylase OTUD3 regulates PTEN stability and suppresses tumorigenesis. Nat Cell Biol 17, 1169–1181 (2015). https://doi.org/10.1038/ncb3218

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