Fig 2: Stabilization of XIAP by USP11 inhibits anoikis and promotes tumor transformation and drug resistance.(A, B) Elevated expression of USP11 inhibits anoikis in MCF10A. (A) Elevated expression of USP11-WT but not C318A significantly decreased activation of Caspase-3, -9 and cleavage of PARP1 in MCF10A cells. (B) The annexin-V-PI staining indicates the elevated USP11-WT but not USP11-C318A decreases anoikis in MCF10A cells. (C, D) Depletion of XIAP decreases USP11 dependent MCF10A transformation. (E) Elevated expression of XIAP-WT but not XIAP-L207P enhances MCF10A transformation. (F) Knockdown of USP11 in MDA-MB-231 cells promotes cell apoptosis in the presence of cisplatin treatment. PARP1 cleavage is marker for apoptosis. (G) Inhibition of USP11 by its inhibitor Mitoxantrone results in sensitizing MDA-MB-231 cells to cisplatin. (H) Inhibition of XIAP by its inhibitor Embelin leads to sensitizing MDA-MB-231 cells to cisplatin. (I, J, K) An in vivo mouse xenograft study was conducted to validate the oncogenic role of USP11 in breast tumorigenesis and tumor progression. (I) 1 × 107 MCF10A cells with stable expression of USP11-WT or USP11-C318A were implanted into right mammary fat pad of SCID mice, the equal amount control MCF10A cells were implanted into left mammary fat pad of SCID mice. Tumor volume was measured once a week for 28 days after injection. Only the wild-type USP11 overexpression but not USP11 death mutant expression can drive MCF10A cells to form tumor after 28 days implanted. (J, K) MDA-MB-231 cells with stable expression of USP11-WT or USP11-C318A were implanted into mammary fat pad of nude mice. Tumor volume was measured once a week and mice were sacrificed 42 days after injection. (K) Lower panel, representative mice show tumor formation. Upper panel, tumor weight was measured. The results were presented as mean values ± s.e.m. *p < 0.01 using Student's t-test.

Fig 3: Abnormal USP11 expression correlates with breast cancer prognosis.(A) Expression of USP11 and XIAP in mammary gland epithelial cell and various types of breast cancer cell lines. While USP11 expression is relatively lower in mammary epithelial cell and moderately in Her2 positive breast cancer cells, significant accumulation of USP11 is observed in triple negative and ER positive type of breast cancer cell lines, including MDA-MB-231, MDA-MB-468, MCF7 and T47D. The down panel showed the statistical analysis of western blotting staining of USP11 expression positively correlated with XIAP expression in breast cancer cell line (R = 0.810, p = 0.0556). (B) Tissue arrays of 65 breast invasive ductal carcinoma and 48 adjacent normal tissue specimens were subjected to immunohistochemistry with anti-USP11 and visualized by DAB staining. Representative normal and cancer tissue staining are shown. (C) Summary of B. (D) Representative staining of well-moderate (W-M) (n = 26) and poor (P) (n = 37) differentiation were shown. (E) Summary of D. (F) Summary of USP11 expression in lymph node negative (LN −) (n = 35) and positive (LN +) (n = 30) breast cancer tissue. (G and H) Kaplan-Meier survival assay of USP11 in overall breast cancer G and ER positive breast cancer H. Patients with USP11 overexpression shows short distant metastasis-free survival (DMSF) time in comparison with patients with low USP11 in either overall breast cancer patients or ER positive patient.

Fig 4: Mapping of the binding interface between USP11 and XIAP.(A) Schematic diagram of human USP11 domains and strategy for engineering of a series of USP11 deletion mutants. (B) Identification of amino acid stretch 503–963 on the carboxyl-terminus of USP11 involved in mediating its interaction with XIAP. (C) Schematic diagram of human XIAP domains and strategy for engineering of a series of XIAP deletion mutants. (D) Identification of molecular region on XIAP, amino acid stretch 93–230, facilitating the interaction between XIAP and USP11. (E) Fine mapping of amino acid stretch 200–210 on the BIR2 domain of XIAP in mediating its interaction with USP11.

Fig 5: USP11 as a deubiquitinase stabilizes XIAP from ubiquitin-dependent degradation.(A) While elevated expression of USP11-WT led to increased XIAP protein levels, expression of deubiquitinase catalytic death mutant USP11 failed to stabilize XIAP in MCF10A cells. (B) Expression of USP11-WT but not USP11-C318A stabilizes Myc tagged XIAP expression levels in HEK293 cells. (C) Depletion of USP11 leads to decreased turnover half-life of XIAP in MDA-MB-231 cells. The density of XIAP band was quantified, normalized to the internal control ß-actin, which are presented in right panel. (D) Elevated expression of USP11 results in prolonged XIAP protein turnover half-life in MCF10A. The density of XIAP band was quantified, normalized to the internal control ß-actin, which are presented in right panel. (E) Blockade of USP11 by pharmacological inhibitor Mitoxantrone destabilizes XIAP protein in MDA-MB-231 cells. MDA-MB-231 cells were treated with Mitoxantrone at indicated concentration for 24 h. (F) Expression of USP11-WT but not USP11-C318A results in decreased XIAP ubiquitin-conjugates in HEK293 cell. HEK293T cells were transfected with the indicated plasmids for 48 h and then collected for immunoprecipitation assay with the FLAG tag antibody followed by the immunoblotting analysis with the HA and Myc tag antibody. (G and H) Elevated expression of USP11 and XIAP are significantly correlated in 65 cases human breast cancer tissue specimen. G Represents paired immunohistochemistry staining of USP11 and XIAP. H Shows statistical analysis of immunohistochemistry staining and indicates USP11 expression is positively correlated with XIAP expression in breast cancer (R = 0.434, p < 0.001).