Journal: Molecular & Cellular Proteomics

Article Title: Elevated Hexokinase II Expression Confers Acquired Resistance to 4-Hydroxytamoxifen in Breast Cancer Cells

doi: 10.1074/mcp.ra119.001576

Figure Lengend Snippet: FIG. 1. LC-PRM for interrogating the differential expression of kinase proteins in tamoxifen-resistant and parental MCF-7 cells. A, A schematic diagram showing the targeted proteomics workflow for human kinome analysis, and depicted here is the forward SILAC labeling experiment. B, Representative PRM traces for mTOR and HK2 proteins. C, Correlation between the ratios obtained from LC-PRM analysis together with forward and reverse SILAC labeling experiments.

Article Snippet: HK2 plasmid was cloned into the NheI and EcoR I restriction sites of the pLJM1-EGFP vector (Addgene, plasmid # 19319).

Techniques: Quantitative Proteomics, Targeted Proteomics, Multiplex sample analysis, Labeling

Journal: Molecular & Cellular Proteomics

Article Title: Elevated Hexokinase II Expression Confers Acquired Resistance to 4-Hydroxytamoxifen in Breast Cancer Cells

doi: 10.1074/mcp.ra119.001576

Figure Lengend Snippet: FIG. 3. TamR MCF-7 cells display elevated expression of HK2 and augmented rate of glycolysis, and elevated expression of HK2 is correlated with poorer survival of breast cancer patients. A, Western blot analysis confirmed the increased expression of HK2 protein in TamR MCF-7 cells. B, Extracellular acidification rates (ECARs) of TamR and parental MCF-7 cells before and after administration of oligomycin and with or without 4-OHT treatment. C, Basal ECAR of TamR cells and parental MCF-7 cells with or without 4-OHT treatment. D, Post-oligomycin maximal ECARs of TamR cells and parental MCF-7 cells with or without 4-OHT treatment. E, Kaplan-Meier survival analysis revealed a significant correlation between higher level of mRNA expression of HK2 gene and poorer relapse-free survival (RFS) in a large cohort of breast cancer patients (n 3951). F, Kaplan-Meier survival analysis showed a significant correlation between higher HK2 mRNA expression and poorer RFS rate in a subgroup of breast cancer patients who received only tamoxifen treatment (n 809). The data represent the mean S.D. of the quantification results (n 3). The p values were calculated based on unpaired, two-tailed Student’s t test: *, 0.01 p 0.05; ***, p 0.001.

Article Snippet: HK2 plasmid was cloned into the NheI and EcoR I restriction sites of the pLJM1-EGFP vector (Addgene, plasmid # 19319).

Techniques: Expressing, Western Blot, Two Tailed Test

Journal: Molecular & Cellular Proteomics

Article Title: Elevated Hexokinase II Expression Confers Acquired Resistance to 4-Hydroxytamoxifen in Breast Cancer Cells

doi: 10.1074/mcp.ra119.001576

Figure Lengend Snippet: FIG. 4. 2-DG-mediated inhibition of HK2 could reduce autophagy induction by 4-OHT and re-sensitize TamR MCF-7 cells to 4-OHT. A, Parental and TamR MCF-7 cells were treated with 2 M 4-OHT for 24 h. At the end of the incubation, 20 nM bafilomycin A1 was added, and after an additional 1 h, autophagy flux was measured, where the expression of LC3B was monitored by Western blotting. B, MCF-7 and TamR MCF-7 cells were treated with 2 M 2-DG for 24 h, and the lysates were used to monitor the expression level of HK2 protein and autophagy marker LC3B by Western blotting. C, D, The ratios of LC3B-II/LC3B-I were quantified from band intensity using ImageJ and the results in D are displayed relative to the level in parental MCF-7 cells without 2-DG treatment. The data were presented as the mean S.D. (n 3). The p values were calculated using unpaired, two-tailed Student’s t test: *, 0.01 p 0.05; **, 0.001 p 0.01; ***, p 0.001. E, F, Parental and TamR MCF-7 cells were treated with serially diluted 4-OHT and 20 nM bafilomycin A1 or 1 mM 2-DG for 72 h, and cell viability was measured using the CCK8 and luminescence was monitored with a BioTek reader. Data were normalized to control groups (DMSO) and represented by the mean and S.D. of results from three independent measurements.

Article Snippet: HK2 plasmid was cloned into the NheI and EcoR I restriction sites of the pLJM1-EGFP vector (Addgene, plasmid # 19319).

Techniques: Inhibition, Incubation, Expressing, Western Blot, Marker, Two Tailed Test, Control

Journal: Molecular & Cellular Proteomics

Article Title: Elevated Hexokinase II Expression Confers Acquired Resistance to 4-Hydroxytamoxifen in Breast Cancer Cells

doi: 10.1074/mcp.ra119.001576

Figure Lengend Snippet: FIG. 5. HK2 inhibition could restore mTOR activity and inhibit autophagy. A, Western blot analysis confirmed the elevated expression of mTOR and decreased S6K phosphorylation in TamR MCF-7 cells. B, Parental and TamR MCF-7 cells were treated with 2 M 4-OHT for 24 h, and the protein expression levels of HK2, mTOR and phospho-S6K were monitored by Western blotting. C, The cells were treated with 2 M 2-DG for 24 h, and the lysates were used to monitor the levels of HK2, mTOR and phospho-S6K by Western blotting analyses. D–F, The ratio of phospho-S6K over S6K was quantified from band intensity using Image J and the values in D and E are displayed relative to levels observed in the parental MCF-7 cells. The data were presented as mean S.D. (n 3). The p values were calculated based on unpaired, two-tailed Student’s t test: *, 0.01 p 0.05; **, 0.001 p 0.01; ***, p 0.001.

Article Snippet: HK2 plasmid was cloned into the NheI and EcoR I restriction sites of the pLJM1-EGFP vector (Addgene, plasmid # 19319).

Techniques: Inhibition, Activity Assay, Western Blot, Expressing, Phospho-proteomics, Two Tailed Test

Journal: Molecular & Cellular Proteomics

Article Title: Elevated Hexokinase II Expression Confers Acquired Resistance to 4-Hydroxytamoxifen in Breast Cancer Cells

doi: 10.1074/mcp.ra119.001576

Figure Lengend Snippet: FIG. 6. Genetic depletion of HK2 led to increased mTOR activity and attenuated autophagy, and re-sensitized tamoxifen-resistant MCF-7 cells to 4-OHT. A, Validation of HK2 and mTOR expression levels and phospho-S6K, LC3B-II/LC3B-I changes after knocking down HK2 in TamR MCF-7 cells with two different sequences of shRNA. B, C, Quantification of changes in p-S6K and LC3B-II/LC3B-I in TamR MCF-7 cells upon shRNA-mediated knockdown of HK2. The values for p-S6K and LC3B-II were normalized against the band intensities of S6K and LC3B-I, respectively, The data were presented as the mean S.D. (n 3). The p values were calculated using unpaired, two-tailed Student’s t test: **, 0.001 p 0.01; ***, p 0.001. D, TamR MCF-7 shHK2 stable knockdown cells were treated with serially diluted 4-OHT for 72 h and cell viability was measured using CCK8. The data were normalized to control groups (ethanol) and represented by the mean and S.D. of results from three independent measurements. E, TamR MCF-7 cells were collected and cell lysates were immuno-precipitated using an antibody for either HK2 or mTOR, and the captured proteins were eluted and Western blotting was employed to monitor the interaction between HK2 and mTOR.

Article Snippet: HK2 plasmid was cloned into the NheI and EcoR I restriction sites of the pLJM1-EGFP vector (Addgene, plasmid # 19319).

Techniques: Activity Assay, Biomarker Discovery, Expressing, shRNA, Knockdown, Two Tailed Test, Control, Western Blot

Journal: Molecular & Cellular Proteomics

Article Title: Elevated Hexokinase II Expression Confers Acquired Resistance to 4-Hydroxytamoxifen in Breast Cancer Cells

doi: 10.1074/mcp.ra119.001576

Figure Lengend Snippet: FIG. 7. Overexpression of HK2 in MCF-7 cells led to decreased mTOR activity, elevated autophagy, and aug- mented resistance to 4-OHT. A, Im- ages from Western blotting for monitor- ing the changes in levels of HK2, mTOR, phospho-S6K, S6K, LC3B-II/LC3B-I in MCF-7 cells after overexpression of HK2. B–C, The quantification data ob- tained from the Western analyses. The data were presented as the mean S.D. (n 3). The p values were calculated based on unpaired, two-tailed Student’s t test: ***, p 0.001. D, MCF-7 cells with stable overexpression of HK2 were treated with serially diluted 4-OHT for 72 h and cell viability was measured with CCK8. The data were normalized to con- trol groups (ethanol) and represented by the mean and S.D. of results from three independent measurements.

Article Snippet: HK2 plasmid was cloned into the NheI and EcoR I restriction sites of the pLJM1-EGFP vector (Addgene, plasmid # 19319).

Techniques: Over Expression, Activity Assay, Western Blot, Two Tailed Test