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. 2019 Feb 7;10(1):636.
doi: 10.1038/s41467-019-08481-x.

PTEN Self-Regulates Through USP11 via the PI3K-FOXO Pathway to Stabilize Tumor Suppression

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Free PMC article

PTEN Self-Regulates Through USP11 via the PI3K-FOXO Pathway to Stabilize Tumor Suppression

Mi Kyung Park et al. Nat Commun. .
Free PMC article

Abstract

PTEN is a lipid phosphatase that antagonizes the PI3K/AKT pathway and is recognized as a major dose-dependent tumor suppressor. The cellular mechanisms that control PTEN levels therefore offer potential routes to therapy, but these are as yet poorly defined. Here we demonstrate that PTEN plays an unexpected role in regulating its own stability through the transcriptional upregulation of the deubiquitinase USP11 by the PI3K/FOXO pathway, and further show that this feedforward mechanism is implicated in its tumor-suppressive role, as mice lacking Usp11 display increased susceptibility to PTEN-dependent tumor initiation, growth and metastasis. Notably, USP11 is downregulated in cancer patients, and correlates with PTEN expression and FOXO nuclear localization. Our findings therefore demonstrate that PTEN-PI3K-FOXO-USP11 constitute the regulatory feedforward loop that improves the stability and tumor suppressive activity of PTEN.

Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
USP11 reduces PIP3 levels by deubiquitinating and stabilizing PTEN. a The screen scheme for the DUB library. b Validation of an RNAi screen in (a). MEFs expressing two independent Usp11 shRNAs were subjected to AlphaScreen assays (top, left) and immunoblotting (IB) (bottom, left). The levels of PIP3 in MEFs expressing Usp11 shRNA were evaluated using an IF (top, middle) and PIP3 Mass ELISA assays (bottom, middle). Lysates and total RNAs from PTEN-proficient DU145 cells and PTEN-deficient PC3 cells complemented with PTEN expressing USP11 shRNAs were subjected to IB (top, right) and RT-qPCR (bottom, right). Scale bars, 10μm. n = 3. Error bars represent  ±  SEM. p Value was determined by Student’s t test (n.s., non-significant; *p < 0.05; **p < 0.01). c Lysates from DU145 cells were immunoprecipitated (IP) without (Mock) or with anti-PTEN (top) or anti-USP11 (bottom) antibody and subjected to IB. # indicates heavy chain of IgG. d Lysates from 293T cells transfected as indicated and treated with 10 μM MG132 for 4 h were IP with anti-Myc–PTEN, and the resulting immunoprecipitates were subjected to IB. # indicates the heavy chain of IgG. HA-Ub, HA-tagged ubiquitin. e Lysates from DU145 cells overexpressing wild-type (WT) or catalytically inactive C318S (CS) mutant of USP11 were subjected to IB. f Lysates from NB4 cells expressing USP11 shRNA and treated with cycloheximide (CHX, 100 μg ml−1) for the indicated times were subjected to IB (top). PTEN protein levels were quantified by normalizing to the intensity of the actin band (bottom). n = 3. Error bars represent  ±   SEM. p Value was determined by ANOVA (*p < 0.05). g IF analysis of PIP3 in Pten+/+ and Pten-/- MEFs expressing Usp11 shRNAs starved for 8 h and stimulated with 100 nM insulin for 5 min. Arrowheads indicate the accumulation of PIP3 at the leading edges of membrane projections. Scale bars, 10μm. h Lysates from Pten+/+ and Pten-/- MEFs expressing Usp11 shRNAs, starved for 8 h and stimulated with serum for 10 min or 100 nM insulin, 100 ng ml−1 IGF-1, or 25 ng ml−1 EGF for 5 min, were subjected to IB. MEFs, mouse embryonic fibroblasts; RT-qPCR, real-time quantitative reverse transcription PCR
Fig. 2
Fig. 2
Loss of Usp11 increases cell proliferation, motility, and metabolism. a Growth curves of primary wild-type (Usp11+/Y) and Usp11-/Y MEFs. n = 3. b Colony-formation efficiency of primary Usp11+/Y and Usp11-/Y MEFs (left). The number of colonies per well was counted (right). n = 3. c Representative plates stained with crystal violet in transformation assays of Usp11+/Y and Usp11-/Y MEFs with the indicated oncogenes are shown (top). Quantification of the number of transformed foci is also shown (bottom). n = 6. d Representative images of allograft tumors of Usp11+/Y and Usp11-/Y MEFs transformed by E1a + Ha-ras oncogenes (top). Lysates from the tumors were subjected to IB (middle). Tumor volumes at different days were measured (bottom). n = 6, p value was determined by ANOVA (***p < 0.001). e Representative images of wound-healing assays of Usp11+/Y and Usp11-/Y MEFs at time points t = 0, 12, and 24 h in the presence of mitomycin C (5 μg ml−1) after wound introduction (left). The percentage of wound closure at the indicated time points was determined by the ImageJ 1.46r software (right). Scale bars, 100μm. n = 3. f MMP family member gene enrichment signature from (e) by microarray. n = 3. Heatmap colors represent the relative mRNA expression as indicated in the color key. g The rates of glucose uptake, lactate production, and glutamine consumption of Usp11+/Y and Usp11-/Y MEFs were measured and normalized to cell number. n = 3. h The rates of glucose uptake, lactate production, and glutamine consumption of MEFs overexpressing USP11 were measured and normalized to cell number. n = 3. i Cytosolic and plasma membrane fractionation of GLUT1 in MEFs overexpressing USP11 treated with insulin (0.5 μg ml−1) for the indicated times is shown. # indicates nonspecific band. Error bars represent   ±   SEM. p Value was determined by Student’s t test (*p < 0.05; **p < 0.01; ***p < 0.001). IB, immunoblotting; MEFs, mouse embryonic fibroblasts
Fig. 3
Fig. 3
Usp11 ablation promotes tumor growth and metastasis in TRAMP mice. a Lysates from anterior prostate (AP) of 11-week-old Usp11+/Y and Usp11-/Y mice were subjected to immunoblotting. b Prostate tissue weight relative to body weight (BW) of 11-week-old Usp11+/Y (n = 8) and Usp11-/Y (n = 8) mice. VP, ventral prostate; AP, anterior prostate; DLP, dorsal-lateral prostate. c Ki-67-stained sections of anterior prostate lobes isolated from 11-week-old Usp11+/Y and Usp11-/Y mice (left). Quantification of the number of Ki-67-positive cells is shown in right. Green scale bars, 75μm; yellow scale bars, 150μm. d H&E-stained sections of dorsal-lateral prostate lobes isolated from 10-week-old TRAMP;Usp11+/Y (n = 8) and TRAMP;Usp11-/Y (n = 6) (left). Quantification of prostatic intraepithelial neoplasia (PIN) is shown in right. Scale bars, 75μm. e Ki-67-stained sections of anterior and ventral prostate lobes isolated from 10-week-old TRAMP;Usp11+/Y (n = 8) and TRAMP;Usp11-/Y (n = 6) (left). Quantification of the number of Ki-67-positive cells is shown in the right. Scale bars, 75μm. f MRI analysis of 20-week-old TRAMP;Usp11+/Y (n = 6) and TRAMP;Usp11-/Y (n = 6) mice (top), and quantification of prostate tumor volume using an Oxirix Imaging software (bottom, left). Prostate tissue weight of 25-week-old TRAMP;Usp11+/Y and TRAMP;Usp11-/Y mice is also shown (bottom, right). g Sections of prostate isolated from 25-week-old TRAMP;Usp11+/Y and TRAMP;Usp11-/Y mice stained with H&E and anti-SV40 T Ag. Scale bars, 75μm. h Sections of prostate isolated from 25-week-old TRAMP;Usp11+/Y and TRAMP;Usp11-/Y mice stained with H&E and anti-smooth muscle actin (SMA) (left). Percentage of invasive prostate carcinoma (CaP) in TRAMP;Usp11+/Y (n = 6) and TRAMP;Usp11-/Y (n = 6) mice is shown in the right. Scale bars, 75μm. i Sections of lymph nodes isolated from 25-week-old TRAMP;Usp11+/Y and TRAMP;Usp11-/Y mice stained with H&E and anti-SV40 T Ag (left). Percentage of lymph node (LN) metastases in TRAMP;Usp11+/Y (n = 6) and TRAMP;Usp11-/Y (n = 5) mice is shown in the right. Scale bars, 75μm. Error bars represent ±   SEM. p Value was determined by Student’s t test (*p < 0.05; **p < 0.01; ***p < 0.001). H&E, hematoxylin and eosin; MRI, magnetic resonance imaging; TRAMP, transgenic adenocarcinoma mouse prostate
Fig. 4
Fig. 4
USP11 suppresses cancer cell biology in a PTEN-dependent manner. a Growth curves of HAP1 cells knocked out (KO) for USP11 using CRISPR/Cas9 technology expressing wild-type (WT) or catalytically inactive C318S (CS) mutant of USP11. n = 3. **p < 0.01, ***p < 0.001, Cont. vs. USP11WT; $$p < 0.01, $$$p < 0.001, USP11WT vs. USP11CS; #p < 0.05, ##p < 0.01, Cont. vs. USP11CS. b Cell-invasion assays of HAP1 cells KO for USP11 expressing WT or CS mutant of USP11 in the presence of mitomycin C (5 μg ml−1) (top). The number of invaded cells per field was quantified (bottom). n = 3. c The rates of glucose uptake, lactate production, and glutamine consumption of HAP1 cells KO for USP11 expressing WT or CS mutant of USP11 were measured and normalized to cell number. n = 3. d Growth curves of PC3 PTENWT and PC3 PTEN-/- cells expressing USP11. n = 3. *p < 0.05, **p < 0.01, PTENWT; Cont. vs. PTENWT; USP11; n.s., non-significant, PTEN-/-;Cont. vs. PTEN-/-;USP11. e Cell-invasion assays of PC3 PTENWT and PC3 PTEN-/- cells ectopically expressing USP11 in the presence of mitomycin C (5 μg ml−1) (top). The number of invaded cells per field was quantified (bottom). n = 3. f The rates of glucose uptake, lactate production, and glutamine consumption of PC3 PTENWT and PC3 PTEN-/- cells ectopically expressing USP11 were measured and normalized to cell number. n = 3. g Growth curves of PC3 PTENWT and PC3 PTEN-/- cells expressing USP11 shRNA. n = 3. *p < 0.05, ***p < 0.001, PTENWT;shCont. vs. PTENWT;shUSP11; n.s., non-significant, $$$p < 0.001, PTEN-/-;shCont. vs. PTEN-/-;shUSP11. h Cell-invasion assays of PC3 PTENWT and PC3 PTEN-/- cells expressing USP11 shRNA in the presence of mitomycin C (5 μg ml−1) (top). The number of invaded cells per field was quantified (bottom). n = 3. i The rates of glucose uptake, lactate production, and glutamine consumption of PC3 PTENWT and PC3 PTEN-/- cells expressing USP11 shRNA were measured and normalized to cell number. n = 3. Scale bars, 100μm. Error bars represent  ±  SEM. p Value was determined by Student’s t test (n.s., non-significant; *p < 0.05, **p < 0.01, ***p < 0.001)
Fig. 5
Fig. 5
USP11 is downregulated and correlates with PTEN in human cancers. a, b USP11 expression in human prostate (a) and breast (b) cancers using a previously published microarray database. Stephenson’s CaP, prostate carcinoma (ref., n = 97); Taylor’s CaP (GEO: GSE21032, n = 179); Liu’s BCa, breast carcinoma (GEO: GSE22820, n = 176). The line in the middle, upper end, and lower end of the boxplot represents the mean, upper, and lower quartile of the relative mRNA level of all samples, respectively. The lines above and below the box are the maximum and minimum values. Data points beyond the whiskers ( > 1.5 interquartile ranges) are drawn as individual dots. Error bars represent ±  SEM. p Value was determined by Student’s t test (**p < 0.01; ***p < 0.001). c Online analysis of relapse-free survival (RFS) (n = 618) and overall survival (OS) (n = 241) in human basal-type breast cancer patients with high or low USP11 expression. The number of surviving patients at different time points is indicated below the graphs. p Value was determined by log-rank (Mantel–Cox) test. HR, hazard ratio. d Fluorescence in situ hybridization (FISH) analysis of PTEN in human prostate (left, n = 116) and TNBC (right, n = 103) tumor samples. The table indicates the frequency of PTEN immunohistochemical (IHC) status by PTEN FISH status. Correlation between PTEN IHC and PTEN FISH was determined by the PASS Pearson Chi-square test. e IHC analysis of USP11 and PTEN in human prostate (top, n = 99) and TNBC (bottom, n = 86) tumor samples (left). Scale bars, 50μm. Correlation between USP11 and PTEN protein levels was determined by the PASS Pearson Chi-square test (right). r, correlation coefficient. TNBC, triple-negative breast cancer
Fig. 6
Fig. 6
USP11-mediated cell density-dependent PTEN regulation. a Representative images of sparse- and dense-confluent primary MEFs (left). Immunoblotting (IB) of sparse- and dense-confluent primary (middle) or transformed MEFs (right). Scale bars, 50μm. b Total RNAs from (a) were subjected to RT-qPCR. n = 3. c Lysates from sparse- and dense-confluent MEFs treated with cycloheximide (CHX, 100 μg ml−1) for the indicated times were subjected to IB (top). PTEN protein levels were quantified by normalizing to the intensity of the actin band (bottom). n = 3, p value was determined by ANOVA. d Lysates from sparse- and dense-confluent MEFs treated with 10 μM MG132 for 4 h were immunoprecipitated (IP) with anti-PTEN, and the resulting IP were subjected to IB. # indicates the heavy chain of IgG. e, f Lysates and total RNAs from sparse- and dense-confluent MEFs were subjected to IB for the indicated proteins (e) and RT-qPCR (f). n = 3. g Lysates from sparse- and dense-confluent MEFs expressing Usp11 shRNA were subjected to IB. h Lysates from sparse- and dense-confluent MEFs expressing two independent Foxo1 shRNAs were subjected to IB. i Luciferase reporter analysis of the USP11 promoter in NIH-3T3 cells expressing Foxo1 shRNAs. n = 3. j Luciferase reporter analysis of two potent FOXO-binding site mutants (Mut) of the USP11 promoter in NIH-3T3 cells expressing FOXO1. n = 3. k Chromatin levels of FOXO1 at the proximal promoters of mouse Usp11 were compared between sparse- and dense-confluent MEFs by quantitative ChIP assays. Enrichment of FOXO1 was analyzed with respect to the input control (before IP) and normalized to IgG control. TSS, transcription start site. n = 3. l Lysates from sparse- and dense-confluent Foxo1+/+ and Foxo1-/- MEFs were subjected to IB. Error bars represent  ±  SEM. p Value was determined by Student’s t test (n.s., non-significant; *p < 0.05, **p < 0.01, ***p < 0.001). ChIP, chromatin immunoprecipitation; MEFs, mouse embryonic fibroblasts; RT-qPCR, quantitative reverse transcription PCR
Fig. 7
Fig. 7
FOXO activates the expression of USP11, thereby upregulating PTEN. a Lysates from DU145 cells expressing SIRT1 shRNA treated with 25 μM resveratrol for 16 h were immunoprecipitated (IP) with anti-FOXO1, and the resulting immunoprecipitates were subjected to immunoblotting (IB). b, c Lysates from DU145 cells expressing two independent shRNAs against SIRT1 (b) or FOXO1 (c) treated with 25 μM resveratrol for 16 h were subjected to IB. d Lysates from DU145 cells pre-treated with 25 μM resveratrol for 16 h followed by treatment with cycloheximide (CHX, 100 μg ml−1) for the indicated times were subjected to IB. n = 3, p value was determined by ANOVA. e Lysates from DU145 cells expressing USP11 shRNA treated with 25 μM resveratrol for 16 h were subjected to IB. f Lysates from DU145 cells expressing USP11 shRNA and treated with 25 μM resveratrol (Resv.) for 16 h and 10 μM MG132 for the last 4 h before harvesting were immunoprecipitated with anti-PTEN, and the resulting immunoprecipitates were subjected to IB. g Luciferase reporter analysis of the USP11 promoter in NIH-3T3 cells expressing constitutive, active FOXO1T24A,T256A,S319A or FOXO3T32A,S253A,S315A. n = 3. h Immunofluorescence analysis of FOXO1 in PTEN-/- HCT116 cells treated with 5 μM psammaplysene A (PsA) for 24 h. Scale bars, 10μm. i Lysates from PTEN+/+ HCT116 cells treated with 5 μM PsA for 24 h were subjected to IB. j Immunohistochemical analysis of FOXO3 or FOXO1 and USP11 in human prostate (left, n = 37) and TNBC (right, n = 68) tumor samples. Scale bars, 50μm. Correlation between FOXO localization and USP11 protein levels was determined by the PASS Pearson Chi-Square test (right). Error bars represent ± SEM. p Value was determined by Student’s t test (*p < 0.05, **p < 0.01, ***p < 0.001). TNBC, triple-negative breast cancer
Fig. 8
Fig. 8
A PTEN-PI3K-FOXO-USP11 auto-regulatory feedforward mechanism. a, b Lysates and total RNAs from PTEN+/+, PTEN+/-, and PTEN-/- HCT116 cells were subjected to immunoblotting (IB) (a) and RT-qPCR (b). n = 3, p value was determined by ANOVA. (c) Luciferase reporter analysis of the USP11 promoter in PTEN+/+, PTEN+/-, and PTEN-/- HCT116 cells. n = 3, p value was determined by ANOVA. d Chromatin levels of FOXO1 at proximal promoters of human USP11 were compared between PTEN+/+ and PTEN-/- HCT116 cells by quantitative ChIP assays. Enrichment of FOXO1 was analyzed with respect to the input control (before IP) and normalized to IgG control. TSS, transcription start site. n = 3. e Lysates and total RNAs from anterior prostates of wild-type (Ptenpc+/+) and Probasin-Cre4;PtenloxP/loxP (Ptenpc-/-) mice at 11 weeks of age (n= 4) were subjected to IB (top) and RT-qPCR (bottom). f Lysates and total RNAs from PTEN+/+, PTEN+/-, and PTEN-/- HCT116 cells treated with 500 nM BKM120 for 24 h were subjected to IB (top) and RT-qPCR (bottom). n = 3. g Lysates and total RNAs from PTEN+/+, PTEN+/-, and PTEN-/- HCT116 cells treated with 500 nM VO-OHpic for 8 h were subjected to IB (top) and RT-qPCR (bottom). n = 3. h Lysates from PTEN+/+ HCT116 cells treated with 500 nM BKM120 for 24 h together with the absence or presence of MG132 (10 μM, 8 h before harvesting) were subjected to IB. i Lysates from p85+/+ and p85-/- MEFs treated with 10 μM MG132 for 8 h were subjected to IB. j Lysates from PTEN-/- HCT116 cells transfected with wild-type (WT) or phosphatase-inactive PTENC124S and PTENG129E subjected to IB. k A model for a PTEN-PI3K-FOXO-USP11 auto-regulatory feedforward mechanism. Error bars represent  ±  SEM. p Value was determined by Student’s t test (n.s. non-significant; *p < 0.05, **p < 0.01, ***p < 0.001). ChIP, chromatin immunoprecipitation; IP, immunoprecipitation; MEFs, mouse embryonic fibroblasts; RT-qPCR, quantitative reverse transcription PCR

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