Fig 2: The interaction between HK2 and CD133 induces binding of USP11 to CD133 and inhibits CD133 polyubiquitylation and degradation. (A) SCLC monolayer or spheroid cells were lysed. Immunoprecipitation and immunoblotting analyses were performed with the indicated antibodies. (B) 293T cells expressing HA-Ub were transfected with or without a Flag-CD133 plasmid, followed by treatment with MG132 (50 µmol/L) for 8 hours. CD133 purified by immunoprecipitation was incubated with or without bacterially purified GST-USP11. Immunoblotting analyses were performed with the indicated antibodies (upper). The purification efficiency was determined using SDS-PAGE and Coomassie brilliant blue staining (lower). (C) shRNA resistant WT Flag-USP11 or inactive Flag-USP11(C318S) mutant was expressed in H1048 USP11 depletion cells. Immunoblotting analyses were performed with the indicated antibodies. (D) H1048 cells expressing two different USP11 shRNAs or a control shRNA were treated with MG132 (50 µmol/L) for 8 hours. Immunoprecipitation and immunoblotting analyses were performed with the indicated antibodies. (E) Two different USP11 shRNAs or a control shRNA were expressed in H1048 cells. Immunoblotting analyses were performed with the indicated antibodies. (F) H1048 cells expressing two different USP11 shRNAs or a control shRNA were examined using a first and the second passage sphere formation assay. Representative images were displayed (upper). The diameters and number of spheres were determined (lower). Data shown are the mean ± SD (n = 3). ***, P < 0.001. Scale bars: 100 µm. (G) 293T cells were transfected with or without Flag-CD133. Purified CD133 was incubated with or without His-HK2, followed by incubation with purified GST-USP11. GST pulldown assay and immunoblotting analyses were performed (upper). The purification efficiency was determined using SDS-PAGE and coomassie brilliant blue staining (lower). (H) H1048 CSCs with endogenous HK2 depletion and with or without reconstituted expression of RNAi-resistant WT Flag-rHK2 or an inactive Flag-rHK2 D209/657A mutant were analyzed by immunoprecipitation and immunoblotting with the indicated antibodies. (I) RNAi-resistant WT HK2, inactive HK2 D209/657A, or HK2 ?N16 was expressed in H1048 cells with depletion of endogenous HK2. Immunoprecipitation and immunoblotting analyses were performed with the indicated antibodies. Abbreviations: HK2, hexokinase 2; USP11, ubiquitin-specific protease 11; SCLC, small cell lung cancer; SDS-PAGE; sodium dodecyl sulfate polyacrylamide gel electrophoresis; shRNA, short hairpin RNA; SD, standard deviation; WT, wide type; IP, immunoprecipitation; WCL, whole cell lysate

Fig 3: A schematic displays the mechanism underlying HK2-regulated cancer cell stemness. In cancer stem cells, HK2 expression is increased. Non-mitochondrial HK2 binds to CD133 and subsequently promotes the interaction between USP11 and CD133, leading to inhibition of CD133 polyubiquitlytion and increase of CD133 stability and CD133 maintenance (left). In differentiated cancer cells, HK2 binds to outer membrane of mitochondria, promoting ubiquitin-proteasome system-mediated CD133 degradation. The light green parts represent the cytoplasm. The red solid arrows represent positive regulation or increased expression of the indicated factors. Black solid arrows represent decrease of gene expression. The black dotted line indicates that the regulation will not occur

Fig 4: HK2 interacts with CD133 and inhibits polyubiquitylation-dependent CD133 degradation. (A-B) HK2 was depleted (A) or overexpressed (B) in H1048 or H69 cells. Immunoblotting analyses were performed with an anti-CD133 antibody. (C-D) H1048 cells with or without HK2 depletion (C) or overexpression (D) were treated with CHX (100 µg/mL) for the indicated periods of time. Immunoblotting analyses were performed with the indicated antibodies (left). Quantification of CD133 expression levels relative to tubulin expression levels is shown (right). (E-F) H1048 cells with or without HK2 depletion (E) or overexpression (F) were treated with MG132 (50 µmol/L) for 8 hours. Immunoprecipitation with an anti-CD133 antibody and immunoblotting analyses were performed with the indicated antibodies. (G) Purified His-HK2 was incubated with purified GST or GST-CD133 together with glutathione-sepharose 4B beads. GST pulldown assay and immunoblotting analyses were performed with the indicated antibodies. (H) Mitochondrial and non-mitochondrial fractions of H1048 monolayers or spheroids were prepared and subjected to immunoprecipitation with an anti-HK2 antibody. Immunoblotting analyses were performed with the indicated antibodies. (I) RNAi-resistant WT HK2, inactive HK2 D209/657A, or HK2 ?N16 was expressed in H1048 and H69 cells with depletion of endogenous HK2. Immunoblotting analyses were performed with the indicated antibodies. (J) H1048 cells with or without WT HK2 or inactive HK2 D209/657A overexpression were treated with MG132 (50 µmol/L) for 8 hours. Immunoprecipitation and immunoblotting analyses were performed with the indicated antibodies. (K) H1048 cells with or without expression of inactive HK2 D209/657A were treated with CHX (100 µg/mL) for the indicated periods of time. Immunoblotting analyses were performed with the indicated antibodies (upper). Quantification of CD133 expression levels relative to tubulin expression levels is shown (lower). Abbreviations: HK2, hexokinase 2; CHX, cycloheximide; IP, immunoprecipitation; WCL, whole cell lysate; WT, wide type

Fig 5: HK2 promotes cancer stem-like properties via CD133. (A) H1048 cells with or without the indicated expression of Flag-HK2, USP11 shRNA, and CD133 were analyzed via immunoblotting with the indicated antibodies. (B) H1048 cells with or without expression of HK2 shRNA and Flag-CD133 were analyzed via immunoblotting with the indicated antibodies. (C) H1048 cells with or without the indicated combined expression of Flag-HK2, USP11 shRNA, and CD133 were subcutaneously injected into the flank regions of nude mice. Tumor sizes (upper) and volumes (lower) were measured and calculated. Data represent the means ± SD of five mice per group. **, P < 0.01, ***; P < 0.001. (D) H1048 cells with or without HK2 shRNA expression or combined expression of HK2 shRNA and Flag-CD133 were subcutaneously injected into the flank regions of nude mice. Tumor sizes (top) and volumes (bottom) were measured and calculated. Data represent the means ± SD of eight mice per group. ***, P < 0.001. (E-F) IHC staining of tumor tissues was performed with the indicated antibodies. Representative images were shown (left or upper). IHC scores (right or lower) were calculated. Data represent the means ± SD (n = 5). *, P < 0.05; ***, P < 0.001. Scale bars: 100 µm. (G) Correlation between HK2 and CD133 expression in an SCLC tissue microarray was analyzed using a two-tailed Pearson correlation coefficient. (H) Representative and magnified IHC images of CD133 protein expression (left) in paired SCLC tumor and adjacent normal tissues from a tissue array were shown. Analysis of the IHC scores of CD133 expression was performed (right). N, adjacent normal tissue; T, tumor tissue. Scale bars: 50 µm. (I) Kaplan–Meier survival curves were compared using a log-rank test in 90 patients. The expression of CD133 was classified as low (score = 4) or high (score >4). Abbreviations: HK2, hexokinase 2; USP11, ubiquitin-specific protease 11; shRNA, short hairpin RNA; SCLC, small cell lung cancer; SD, standard deviation; IHC, immunohistochemistry