. 2025 Sep 23;135(22):e180927.

doi: 10.1172/JCI180927. eCollection 2025 Nov 17.

USP10/GSK3β-mediated inhibition of PTEN drives resistance to PI3K inhibitors in breast cancer

Nishi Kumari¹, Sarah Ce Wright^{2

3}, Christopher M Witham^{2

3}, Laia Monserrat⁴, Marta Palafox⁴, John Lc Richard¹, Carlotta Costa⁵, Moshe Elkabets⁶, Mark Agostino^{2

3}, Theresa Klemm⁷, Melissa Eccles^{2

3}, Alex Garnham^{7

8}, Ting Wu⁹, Jonas A Nilsson⁹, Nikita Walz^{2

3}, Veena Venugopal¹, Anthony Cerra⁷, Natali Vasilevski^{2

3}, Stephanie Bridgeman^{2

3}, Sona Bassi^{2

3}, Azad Saei^{1

10

11}, Moutaz Helal¹², Philipp Neundorf¹², Angela Riedel¹², Mathias Rosenfeldt^{12

13}, Jespal Gill¹⁴, Nikolett Pahor^{15

16}, Oliver Hartmann^{15

16}, Jacky Chung¹⁷, Sachdev S Sidhu¹⁷, Nina Moderau¹⁸, Sudhakar Jha¹⁹, Jordi Rodon²⁰, Markus E Diefenbacher^{15

16

21}, David Komander^{7

22}, Violeta Serra⁴, Pieter Johan Adam Eichhorn^{2

3}

Affiliations

¹ Cancer Science Institute of Singapore, National University of Singapore, Singapore.
² Curtin Medical School, Faculty of Health Sciences and.
³ Curtin Medical Research Institute, Curtin University, Bentley, Western Australia, Australia.
⁴ Vall d'Hebron Institute of Oncology, Barcelona, Spain.
⁵ Massachusetts General Hospital Cancer Center and Harvard Medical School, Boston, Massachusetts, USA.
⁶ The Shraga Segal Department of Microbiology, Immunology, and Genetics, Ben-Gurion University of the Negev, Beer-Sheva, Israel.
⁷ The Walter and Eliza Hall Institute of Medical Research, Melbourne, Victoria, Australia.
⁸ Department of Medical Biology, The University of Melbourne, Parkville, Victoria, Australia.
⁹ Harry Perkins Institute of Medical Research, University of Western Australia, Perth, Western Australia, Australia.
¹⁰ Department of Pharmacology, Yong Loo Lin School of Medicine National University of Singapore, Singapore.
¹¹ Genome Institute of Singapore, A*STAR, Singapore, Singapore.
¹² Mildred Scheel Early Career Centre, University Hospital Wuerzburg, Wuerzburg, Germany.
¹³ Institute for Pathology, University of Wuerzburg, Wuerzburg, Germany.
¹⁴ Anatomical Pathology, Western Diagnostics, Murdoch, Western Australia, Australia.
¹⁵ Institute of Lung Health and Immunity, Helmholtz Center, Munich, Germany.
¹⁶ German Center for Lung Research, Munich, Germany.
¹⁷ School of Pharmacy, University of Waterloo, Kitchener, Ontario, Canada.
¹⁸ Department of Surgery and Cancer, Faculty of Medicine, Imperial College London, London, United Kingdom.
¹⁹ Department of Physiological Sciences, College of Veterinary Medicine, Oklahoma State University, Stillwater, Oklahoma, USA.
²⁰ MD Anderson Cancer Center, Investigational Cancer Therapeutics Department, Houston, Texas, USA.
²¹ Ludwig Maximilian University, Munich, Germany.
²² Department of Medical Biology, University of Melbourne, Royal Parade, Melbourne, Victoria, Australia.

PMID: 40991650
PMCID: PMC12618074
DOI: 10.1172/JCI180927

USP10/GSK3β-mediated inhibition of PTEN drives resistance to PI3K inhibitors in breast cancer

Nishi Kumari et al. J Clin Invest. 2025.

. 2025 Sep 23;135(22):e180927.

doi: 10.1172/JCI180927. eCollection 2025 Nov 17.

Authors

Affiliations

¹ Cancer Science Institute of Singapore, National University of Singapore, Singapore.
² Curtin Medical School, Faculty of Health Sciences and.
³ Curtin Medical Research Institute, Curtin University, Bentley, Western Australia, Australia.
⁴ Vall d'Hebron Institute of Oncology, Barcelona, Spain.
⁵ Massachusetts General Hospital Cancer Center and Harvard Medical School, Boston, Massachusetts, USA.
⁶ The Shraga Segal Department of Microbiology, Immunology, and Genetics, Ben-Gurion University of the Negev, Beer-Sheva, Israel.
⁷ The Walter and Eliza Hall Institute of Medical Research, Melbourne, Victoria, Australia.
⁸ Department of Medical Biology, The University of Melbourne, Parkville, Victoria, Australia.
⁹ Harry Perkins Institute of Medical Research, University of Western Australia, Perth, Western Australia, Australia.
¹⁰ Department of Pharmacology, Yong Loo Lin School of Medicine National University of Singapore, Singapore.
¹¹ Genome Institute of Singapore, A*STAR, Singapore, Singapore.
¹² Mildred Scheel Early Career Centre, University Hospital Wuerzburg, Wuerzburg, Germany.
¹³ Institute for Pathology, University of Wuerzburg, Wuerzburg, Germany.
¹⁴ Anatomical Pathology, Western Diagnostics, Murdoch, Western Australia, Australia.
¹⁵ Institute of Lung Health and Immunity, Helmholtz Center, Munich, Germany.
¹⁶ German Center for Lung Research, Munich, Germany.
¹⁷ School of Pharmacy, University of Waterloo, Kitchener, Ontario, Canada.
¹⁸ Department of Surgery and Cancer, Faculty of Medicine, Imperial College London, London, United Kingdom.
¹⁹ Department of Physiological Sciences, College of Veterinary Medicine, Oklahoma State University, Stillwater, Oklahoma, USA.
²⁰ MD Anderson Cancer Center, Investigational Cancer Therapeutics Department, Houston, Texas, USA.
²¹ Ludwig Maximilian University, Munich, Germany.
²² Department of Medical Biology, University of Melbourne, Royal Parade, Melbourne, Victoria, Australia.

PMID: 40991650
PMCID: PMC12618074
DOI: 10.1172/JCI180927

Abstract

Activating mutations in PIK3CA, the gene encoding the catalytic p110α subunit of PI3K, are some of the most frequent genomic alterations in breast cancer. Alpelisib, a small-molecule inhibitor that targets p110α, is a recommended drug for patients with PIK3CA-mutant advanced breast cancer. However, clinical success for PI3K inhibitors (PI3Kis) has been limited by their narrow therapeutic window. The lipid phosphatase PTEN is a potent tumor suppressor and a major negative regulator of the PI3K pathway. Unsurprisingly, inactivating mutations in PTEN correlate with tumor progression and resistance to PI3K inhibition due to persistent PI3K signaling. Here, we demonstrate that PI3K inhibition leads rapidly to the inactivation of PTEN. Using a functional genetic screen, we show that this effect is mediated by a USP10-GSK3β signaling axis, in which USP10 stabilizes GSK3β, resulting in GSK3β-mediated phosphorylation of the C-terminal tail of PTEN. This phosphorylation inhibits PTEN dimerization and thus prevents its activation. Downregulation of GSK3β or USP10 resensitizes PI3Ki-resistant breast cancer models and patient-derived organoids to PI3K inhibition and induces tumor regression. Our study establishes that enhancing PTEN activity is a new strategy to treat PIK3CA mutant tumors and provides a strong rationale for pursuing USP10 inhibitors in the clinic.

Keywords: Breast cancer; Cell biology; Oncology; Signal transduction; Ubiquitin-proteosome system.

PubMed Disclaimer

Figures

**Figure 1. PI3K inhibitor monotherapy in PI3K mutant breast cancers.**
(A) Waterfall plot representing the growth of 27 PDXs treated with BYL719 (35 mg/kg) or GDC-0032 (25 mg/kg). The number of tumors (black circles) treated per model is indicated in the brackets (n). Bars represent the average of treated samples. Vehicle control average is represented as white circles. The percentage change from the initial volume is shown at day 35 of treatment. Dashed lines indicate the range of PD (>20%), SD (20% to –30%) and PR/CR (<–30%). Data are represented as mean values ± SEM. (B) Whole-cell lysates of ER⁺ patient derived xenografts derived from A were probed with indicated antibodies.

**Figure 2. USP10 is a regulator of PI3K signaling in breast cancer.**
(A) Immunoblot analysis of HEK293T cells expressing shRNA vectors targeting the indicated DUBs. Ctl, control. (B) Immunoblot analysis of HEK293T cells expressing USP10 shRNA vectors A and B and probed with the indicated antibodies. (C) Immunoblot analysis in HEK293T cells transfected as indicated and probed with the indicated antibodies. (D) Immunoblot analysis of MCF7 cells expressing USP10 shRNA vectors L1 and L2 probed with the indicated antibodies. Mut, mutation. (E) T47D cells expressing USP10 shRNA vectors L1 and L2, treated with IGF (300 ng/mL) as indicated. Lysates were probed with the indicated antibodies. (F and G) Immunoblot analysis of HEK293T (F) or MCF7 cells (G) or USP10 KO counterparts (USP10^KO1). Lysates were probed with the indicated antibodies. (H) Overview H&E image of human breast cancer FFPE sample used for spatial transcriptomic analysis. (https://www.10xgenomics.com/datasets/human-breast-cancer-ductal-carcinoma-in-situ-invasive-carcinoma-ffpe-1-standard-1-3-0). Data were visualized using the 10x Genomics Loupe browser (version 8.0). (I) K means = 2 clustering of human breast cancer FFPE sample used to segment tumor (BRCA indicated by orange) and stromal tissue (stroma indicated by black). (J) Uniform Manifold Approximation and Projection (UMAP) analysis of cell clustering of the 2 identified clusters displaying genetically defined subclones/differential gene expression of BRCA. (K) Correlation analysis of USP10 and PIK-AKT-Hallmark gene set (HALLMARK_PI3K_AKT_MTOR-SIGNLING, M5923) in UMAP on the breast cancer tissue section. USP10 and PIK-AKT signature-only expression areas are represented in yellow and blue, respectively; co-expression is visualized in green. (L) Co-expression of USP10 and the PIK-AKT signature from UMAP (J). Inset demonstrates co-expression of USP10 and PIK-AKT-Hallmark genes in BRCA. (M) Lysates of HEK293T cells expressing recombinant UbV10 incubated with HA-tagged Ub-VS for 30 minutes. Lysates were probed with a USP10 antibody. (N) MCF7 cells expressing ubiquitin binding variant–USP10 or luciferase control, as indicated. Lysates were probed with indicated antibodies. (O) Phospholipids were isolated from cells treated with DMSO or IGF (300 ng/mL) for 1 hour, and relative PIP3 and PI(4,5)P₂ levels were quantified by ELISA. *P < 0.05 by 2-tailed t test.

**Figure 3. USP10 binds PTEN and regulates its protein expression.**
(A) Whole-cell lysates of PDXs from Figure 1B were probed with indicated antibodies. (B) Colony formation assay of MCF7 or T47D or corresponding PI3Ki-resistant clones R¹, R², and R³ treated with BYL719 (1 μM) or GDC0941 (1 μM) for 21 days. (C and D) Immunoblot analysis of MCF7 cells (C) or T47D cells (D) and corresponding resistant clones. Lysates were probed with the indicated antibodies. (E) HEK293T cells were lysed and immunoprecipitated with a PTEN antibody or IgG control (Ctl). Immunoprecipitated lysates and whole-cell extracts were probed with the indicated antibodies. (F) HEK293T cells were transfected with MYC-tagged PTEN and FLAG-tagged USP10 or FLAG-tagged USP10DD. After 48 hours, cells were lysed and blotted precipitates were probed with the indicated antibodies. (G) HEK293T cells were transfected with MYC-tagged PTEN and either USP10 shRNA vectors L1, L2, or L3. After 72 hours, cells were lysed and blotted precipitates were probed with the indicated antibodies. (H) Immunoblot analysis of HEK293T cells or HEK293T USP10 CRISPR KO cells (USP10^KO1). Whole-cell lysates were probed with the indicated antibodies. (I and J) Representative immunohistology staining of tissue microarrays (TMAs) against endogenous USP10 (I) or PTEN (J) in sample and patient-matched nontransformed and human BRCA samples. Scale bar core overview: 1 mm; zoomed in: 50 μm. (K) Quantitative correlative analysis of protein abundance via IHC intensity in human BRCA using USP10 and PTEN in TMA sections. The analysis was conducted with the image analysis software QuPath (0.5.0) and manually by a trained pathologist. Pearson correlation between positive immunohistology signals of USP10 versus PTEN in human BRCA cores. P < 0.05 by 2-tailed t test. (L) HEK293T cells were transfected with increasing concentrations of FLAG-tagged USP10. After 48 hours, cells were lysed and blotted precipitates were probed with the indicated antibodies.

**Figure 4. USP10 expression correlates with upregulation of PTEN and PI3K pathway signaling.**
(A and B) K means = 3 clustering of human invasive ductal carcinoma single-cell sequencing data set (https://www.10xgenomics.com/datasets/7-5-k-sorted-cells-from-human-invasive-ductal-carcinoma-3-v-3-1-3-1-standard-6-0-0). (A) Heatmap of differentially expressed genes in the K-means = 3 clusters. (B) USP10 expression in the different clusters is shown (black). Cluster 1 = cancer, cluster 2 = unidentified/mixed tissue, cluster 3 = stroma. (C) Violin plots of USP10, PTEN, PCNA, or PIK-AKT Hallmark genes in the K means = 3 clusters. Data demonstrate the predominant expression of USP10 and PCNA in cancer tissue (cluster 1), PTEN expression in cluster 1 (BRCA) and stroma (Cluster 3), and the expression of PIK-AKT hallmark genes in clusters 1 and 3, respectively. Data were visualized using the 10x Genomics Loupe browser (version 8.0). (D) Correlation analysis of the expression of USP10 and PTEN in the single-cell sequencing data set on human invasive ductal carcinoma. Cells with predominantly high expression of USP10 are represented in blue and PTEN-expressing cells are marked in yellow. Co-expression is visualized in green. The zoomed-in view shows the predominant co-expression of USP10 and PTEN in BRCA. Data were visualized using the 10x Genomics Loupe browser (version 8.0). (E) Correlation analysis of USP10 and genes of the PIK-AKT-Hallmark gene set on human invasive ductal carcinoma. Areas of high USP10 expression are represented in yellow, PIK-AKT-Hallmark gene sets only are marked in blue, co-expression is visualized in green. The zoomed-in view shows the predominant co-expression of USP10 and PIK-AKT-Hallmark genes in BRCA. Data were visualized using the 10x Genomics Loupe browser (version 8.0).

**Figure 5. PI3Kis induce PTEN T366 phosphorylation, regulating its dimerization.**
(A) Schematic of PTEN in an open, active, unphosphorylated state (top) and PTEN phosphorylation in the C-terminal tail inducing a closed, inactive conformation (bottom). (B) Co-IP assay from total lysate of HEK293T cells, transfected with GFP-PTEN or FLAG-PTEN, with or without shRNA USP10^L1. IP with anti-FLAG antibody. Western blot was probed with the indicated antibodies. (C) Co-IP assay from total lysate of HEK293T cells or HEK293T USP10^KO1 cells transfected with GFP-PTEN and FLAG-PTEN. IP with anti-FLAG antibody. Western blot was probed with indicated antibodies. (D) Co-IP assay from total lysate of HEK293T, HEK293T USP10^KO1, or HEK293T USP10^KO1 cells ectopically expressing USP10, transfected with GFP-PTEN and FLAG -PTEN. IP with anti-FLAG antibody. Western blot was probed with the indicated antibodies. (E) Initially prepared model of unphosphorylated PTEN dimer or phosphorylated PTEN dimer (pT366) prior to molecular dynamics simulation. Phosphorylated residues are shown as spheres. Blue indicates monomer 1; red indicates monomer 2; teal/magenta indicates phosphothreonine residues. (F) Molecular mechanics — general born surface area binding energies for PTEN dimers with differential phosphorylation status. (G) Co-IP assay from total lysate of HEK293T cells, transfected with GFP-PTEN, HA-PTEN, or HA-PTEN T366A. IP with anti-GFP antibody. Western blot was probed with the indicated antibodies. (H) MCF7 cells treated for 24 hours with 1 μM indicated PI3Kis or AKT inhibitors (AKTi). Whole-cell lysates were collected and probed with the indicated antibodies. (I) MCF7 cells transfected with GFP-PTEN and FLAG-PTEN were treated with 10 μM BYL719, as indicated. IP with anti-FLAG antibody. Western blot was probed with indicated antibodies.

**Figure 6. USP10 stabilizes GSK3β to regulate downstream PI3K signaling.**
(A) Co-IP assay from total lysate of HEK293T cells, transfected with FLAG-GSK3β and/or USP10. IP with anti-FLAG antibody. Western blot was probed with indicated antibodies. (B) Immunoblot analysis of HEK293T cells expressing FLAG-GSK3β with or without shRNA targeting USP10 and probed with the indicated antibodies. Ctl, control. (C) Immunoblot analysis of HEK293T cells or HEK293T USP10 CRISPR KO clones (USP10^KO1, USP10^KO2, USP10^KO3). Whole-cell lysates were probed with the indicated antibodies. (D) Immunoblot analysis in HEK293T cells expressing FLAG-GSK3β, with or without FLAG-USP10 or FLAG-USP10DD. Whole-cell extracts were probed with the indicated antibodies. (E) Immunoblot analysis in HEK293T cells expressing FLAG-GSK3β and either FLAG-USP7, FLAG-USP10, or FLAG-USP13. Whole-cell extracts were probed with the indicated antibodies. (F) Immunoblot analysis in HEK293T cells expressing FLAG-GSK3β and either shRNA USP10^L1 or USP10^L2. Whole-cell extracts were probed with the indicated antibodies. (G) Immunoblot analysis in HEK293T cells expressing FLAG-GSK3β and either shRNA USP10^L1 or USP10^L2. Whole-cell extracts were probed with the indicated antibodies. (H) Co-IP assay from total lysate of HEK293T cells, transfected with GFP-PTEN, HA-PTEN, and HA-GSK3β^Ser9. IP with anti- FLAG antibody. Western blot was probed with indicated antibodies. (I) HEK293T cells transfected with FLAG- GSK3β, HA-tagged ubiquitin, or its mutant isoforms K27, K48, or K63, in the presence or absence of CMV-USP10. Lysates were immunoprecipitated with anti-FLAG affinity resin, resolved by SDS-PAGE, and probed with indicated antibodies.

**Figure 7. USP10 is upregulated in breast cancer and correlates with PI3Ki resistance. (A)**
A batch-corrected Uniform Manifold Approximation and Projection (UMAP), representing malignant and normal epithelial cells from patients with triple-negative breast cancer (53). The cells are colored to depict their respective cell-type annotations: epithelial (green) or malignant (red). (B) A UMAP illustrating the normalized expression of USP10 across all cells extrapolated from A. (C) Matrix heatmap generated using cBioportal showing genetic alterations in PIK3CA, PTEN, and upregulation of USP10 mRNA. (D) Kaplan-Meier curves from TCGA data extrapolated from the GEPIA2 genomics analysis and visualization platform showing probability of overall survival of patients with breast cancer with a higher copy number of USP10 is significantly less than those with a lower level of USP10 (P = 0.049). HR, hazard ratio. (E) MCF7 PI3Ki-resistant cells expressing shRNA USP10L2 or USP10 L3 or shGFP were treated with escalating doses of BYL719, as indicated, for 72 hours. Viability was assayed using CellTiter-Glo as described by the manufacturer. Data represent the mean of 5 replicates. (F) Colony formation assay of MCF7-WT or MCF7-PI3Ki–resistant clone R³ stably expressing shRNA USP10^L1 or USP10^L2 or a shRNA GFP control. Cells were treated with BYL719 (2 μM) for 21 days. (G) Immunoblot analysis of MCF7 or MCF7R3 cells expressing either shRNA USP10^L1 or USP10^L3. Whole-cell extracts were probed with the indicated antibodies. (H) Quantification of the sub-G1 population after treatment with BYL719 (1 μM); the mean SEM of 3 independent experiments is reported. Dunnett’s multiple comparison test was used to compare treated populations. **P < 0.01, ***P < 0.001.

**Figure 8. GSK3β inhibition resensitizes PI3Ki-resistant cells to PI3Kis.**
(A) A dose matrix of BYL719 with 9-ING-41 was created in parental MCF7 cells. Viability was assessed after 5 days. Percentage inhibition at each dose is presented. (B and C) Synergy analysis of BYL719 and 9-ING-41 in an MCF7 cell line showing the ZIP synergy score, as analyzed using SynergyFinder software. (D) A dose matrix of BYL719 with 9-ING-41 was created in MCF7 BYL719-resistant cells. Viability was assessed after 5 days. Percentage inhibition at each dose is presented. Res, resistant. (E and F) Synergy analysis of BYL719 and 9-ING-41 in an MCF7 BYL719-resistant cell line, showing the ZIP synergy score, as analyzed using SynergyFinder software. (G) Colony formation assay of MCF7 or MCF7 BYL719-resistant clones treated with DMSO, BYL719 (1 μM), 9-ING-41 (1 μM), or the combination (Combo) for 21 days. Ctrl, control. (H) Colony formation assay of MCF7 or MCF7 GDC0941-resistant clones treated with DMSO, GDC0941 (1 μM), 9-ING-41 (1 μM), or the combination for 21 days. (I) Viability assay of PDXO479 treated with BYL719 (1 μM), 9-ING-41 (1 μM), or the combination for 5 days. (J) Immunoblot analysis of PDXO479 treated in I. Whole-cell extracts were probed with the indicated antibodies. (K) Schematic overview of GSK3β-USP10 regulation of PTEN dimerization following PI3K inhibition (inh). Mut, mutation.

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USP10/GSK3β-mediated inhibition of PTEN drives resistance to PI3K inhibitors in breast cancer

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USP10/GSK3β-mediated inhibition of PTEN drives resistance to PI3K inhibitors in breast cancer

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